Il-17a-binding polypeptides

ABSTRACT

The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-17A (IL-17A), and provides an IL-17A binding polypeptide comprising the sequence EX2DX4AX6X7EIX10X11LPNL X16X17X18QX20X21AFIX25X26LX28X29. Also disclosed is the use of such an interleukin-17A binding polypeptide as a diagnostic, prognostic and/or therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/538,890 filed Jun. 22, 2017 which is a U.S. National StageApplication of PCT/EP2016/050456 filed Jan. 12, 2016 which claimspriority to EP Application No. 15150786.0 filed Jan. 12, 2015, all ofwhich are incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates to a class of engineered polypeptideshaving a binding affinity for interleukin-17A (in the following referredto as IL-17A). The present disclosure also relates to the use of such aninterleukin-17A binding polypeptide as a diagnostic, prognostic and/ortherapeutic agent.

BACKGROUND

The interleukin-17 (IL-17) family is a pro-inflammatory cytokine familythat contributes to the pathogenesis of several inflammatory diseases. Amajor source of IL-17 is a lineage of T cells known as T helper 17 cells(Th17 cells), which are distinct from the classical Th1 and Th2 cellsubsets. Results of studies in mouse models and in humans haveidentified a key role of IL-17 and Th17 cells in the pathogenesis ofinflammation and autoimmunity as well as in host defense against certainpathogens. Based on these observations, IL-17 and Th17 cells areconsidered to be interesting targets for the treatment of severalchronic inflammatory diseases such as psoriasis, rheumatoid arthritis(RA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE)and multiple sclerosis (MS) (Miossec and Kolls, 2012, Nat Rev DrugDiscov 11:763-7).

The disulfide-linked homodimeric cytokine IL-17A is a member of theIL-17 family, which also includes IL-17B, IL-17C, IL-17D, IL-17E andIL-17F. Within the family, IL-17A and IL-17F show the highest amino acidsequence homology to each other (50%) and they bind to the samereceptors: IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC).Furthermore, IL-17A can be expressed with IL-17F as a heterodimer.Although IL-17A and IL-17F share high amino acid sequence homology, theyperform distinct functions. IL-17A is involved in the development ofautoimmunity, inflammation and tumors and also plays important roles inthe host defense against bacterial and fungal infections. IL-17F, on theother hand, is mainly involved in mucosal host defense mechanisms(Iwakura et al, 2011, Immunity 34:149-62).

When IL-17A is secreted, it promotes the production of a variety ofproinflammatory cytokines, chemokines, antimicrobial peptides andmetalloproteinases (MMPs) from fibroblast, endothelial and epithelialcells. One important action of IL-17A is to induce granulopoiesis andneutrophil recruitment to inflammatory sites. However, if uncontrolled,this reaction may lead to chronic inflammation with tissue destructionand neovascularization (Iwakura et al. 2008, Immunol Rev 226:57-79;Reynolds et al. 2010, Cytokine Growth Factor Rev 21:413-23). IL-17A iscentral in the pathogenesis of psoriasis, a common chronic inflammatoryskin disease affecting about 2.5% of the worldwide population (reviewedin Chiricozzi and Krueger, 2013, Expert Opin. Investig. Drugs22(8):993-1005). Studies in patients with RA have shown that IL-17Apositive cells are present in the inflamed synovium. In a mouse model ofRA, the clinical scores were severely aggravated by administration ofIL-17A via intra-articular gene transfer (Lubberts et al. 2002, InflammRes 51:102-4). Conversely, inhibition of IL-17A with monoclonalantibodies against the ligand or the receptor protected againstdevelopment and consequences of arthritis (Lubberts et al. 2004,Arthritis Rheum 50:650-9). In MS patients, the IL-17A gene is reportedto be overexpressed (Lock et al. 2002, Nat Med 8:500-8) and IL-17A andTh17 cells have been clearly implicated in the mouse model ofexperimental autoimmune encephalitis (Cua et al. 2003, Nature 421:744-8;Uyttenhove and Van Snick 2006, Eur J Immunol 36:2868-74). Increasedlevels of IL-17A have been shown to be clinically correlated withvarious ocular inflammatory diseases, such as uveitis, scleritis and dryeye disease (DED) in patients suffering from arthritis (Kang et al.2011, J Korean Med Sci 26:938-44). Recent studies have showed IL-17 andIFNγ positive cells in clinical specimens of coronary atherosclerosissuggesting a local effect on vessel dysfunction (Eid et al. 2009, JCardiothorac Surg 4:58). IL-17A may also be of interest in chronicobstructive pulmonary disease (COPD). The number of IL-17A positivecells is increased in lung tissues from COPD patients (Chu et al. 2011,Int Immunopharmacol 11:1780-8; Di Stefano et al. 2009, Clin Exp Immunol157:316-24). IL-17RA deficient mice are resistant to the development ofemphysema in a mouse model of COPD whereas overexpression of IL-17Aaccelerates the development of emphysema suggesting that IL-17A issufficient to mediate this response (Chen et al. 2011, PLoS One6:e20333; Shan et al. 2012, Sci Transl Med 4:117ra9). Thus, theinvolvement of IL-17A in several different autoimmune and inflammatorydiseases suggests a wide applicability of therapeutics targeting IL-17A.

Targeting of IL-17A or its receptors is the most direct way to blockIL-17A-mediated functions. Several biologics that neutralize IL-17Asignaling are now in clinical development, including the anti-IL-17Amonoclonal antibodies secukinumab and ixekizumab (Patel et al, 2013, AnnRheum Dis 72 Suppl 2:ii116-23). Secukinumab has been approved for thetreatment of psoriasis and is currently investigated for the treatmentof psoriatic arthritis (PsA) and AS. Ixekizumab is currently in clinicaltrials for psoriasis, PsA and RA. Blocking of IL-17 receptor mediatedsignaling is also under investigation in the clinic, including the humanmonoclonal anti-IL-17RA antibody brodalumab for treatment of psoriasis,RA and asthma (Hu et al. 2011, Ann N Y Acad Sci 1217:60-76). Thus,clinical efficacy of IL-17A-inhibition has been proven in differentdiseases, notably in psoriasis, and the safety profile, including phaseII and phase III data, shows good tolerability for IL-17A inhibitors(Genovese et al. 2010, Arthritis Rheum 62:929-39 and Hueber et al. 2010,Sci Transl Med 2:52ra72).

The unpredictable and chronic nature of psoriasis and other inflammatorydiseases, as well as a high unmet medical need, warrants the developmentof new modes of treatment.

Since tissue penetration rate is negatively associated with the size ofthe molecule, a relatively large antibody molecule inherently has poortissue distribution and penetration capacity. Moreover, althoughantibodies are widely used in a variety of routine contexts owing tohigh affinity and specificity to a multitude of possible antigens, suchas for analytical, purification, diagnostic and therapeutic purposes,they still suffer from several drawbacks. Such drawbacks include theneed for complex mammalian expression systems, aggregation tendencies,limited solubility, need to form and stably maintain disulfide bonds,and the risk of unwanted immune responses.

Thus, the use of monoclonal antibodies is not always optimal fortherapy, and there is continued need for provision of agents with a highaffinity for IL-17A. Of great interest is also the provision of uses ofsuch molecules in the treatment, diagnosis and prognosis of disease.

SUMMARY OF THE INVENTION

It is an object of the present disclosure to provide new IL-17A bindingagents, which could for example be used for diagnostic, prognostic andtherapeutic applications.

It is an object of the present disclosure to provide new IL-17A bindingagents, which may be used as domains in fusion proteins comprising oneor more additional domains having similar or other functions.

It is an object of the present disclosure to provide a molecule allowingfor efficient therapy targeting various forms of inflammatory andautoimmune disease while alleviating the abovementioned and otherdrawbacks of current therapies.

It is a further object of the present disclosure to provide a moleculesuitable for prognostic and diagnostic applications.

These and other objects which are evident to the skilled person from thepresent disclosure are met by different aspects of the invention asclaimed in the appended claims and as generally disclosed herein.

Thus, in the first aspect of the disclosure, there is provided an IL-17Abinding polypeptide, comprising an IL-17A binding motif BM, which motifconsists of an amino acid sequence selected from:

(SEQ ID NO: 1295) EX₂DX₄AX₆X₇EIX₁₀X₁₁LPNLX₁₆X₁₇X₁₈QX₂₀X₂₁AFIX₂₅X₂₆LX₂₈X₂₉wherein, independently from each other,

X₂ is selected from A, H, M and Y;

X₄ is selected from A, D, E, F, K, L, M, N, Q, R, S and Y;

X₆ is selected from A, Q and W;

X₇ is selected from F, I, L, M, V, W and Y;

X₁₀ is selected from A and W;

X₁₁ is selected from A, D, E, F, G, L, M, N, Q, S, T and Y;

X₁₆ is selected from N and T;

X₁₇ is selected from H, W and Y;

X₁₈ is selected from A, D, E, H and V;

X₂₀ is selected from A, G, Q, S and W;

X₂₁ is selected from A, D, E, F, H, K, N, R, T, V, W and Y;

X₂₅ is selected from A, D, E, G, H, I, L, M, N, Q, R, S, T and V;

X₂₆ is selected from K and S;

X₂₈ is selected from I, L, N and R; and

X₂₉ is selected from D and R;

and

-   ii) an amino acid sequence which has at least 89% identity to the    sequence defined in i).

The above definition of a class of sequence related, IL-17A bindingpolypeptides is based on a statistical analysis of a number of randompolypeptide variants of a parent scaffold, that were selected for theirinteraction with IL-17A in several different selection experiments. Theidentified IL-17A binding motif, or “BM”, corresponds to the targetbinding region of the parent scaffold, which region constitutes twoalpha helices within a three-helical bundle protein domain. In theparent scaffold, the varied amino acid residues of the two BM helicesconstitute a binding surface for interaction with the constant Fc partof antibodies. In the present disclosure, the random variation ofbinding surface residues and subsequent selection of variants havereplaced the Fc interaction capacity with a capacity for interactionwith IL-17A.

As the skilled person will realize, the function of any polypeptide,such as the IL-17A binding capacity of the polypeptide of the presentdisclosure, is dependent on the tertiary structure of the polypeptide.It is therefore possible to make minor changes to the sequence of aminoacids in a polypeptide without affecting the function thereof. Thus, thedisclosure encompasses modified variants of the IL-17A bindingpolypeptide, which are such that the IL-17A binding characteristics areretained.

In this way, also encompassed by the present disclosure is an IL-17Abinding polypeptide comprising an amino acid sequence with 89% orgreater identity to a polypeptide as defined in i). In some embodiments,the polypeptide may comprise a sequence which is at least 93%, such asat least 96% identical to a polypeptide as defined in i). For example,it is possible that an amino acid residue belonging to a certainfunctional grouping of amino acid residues (e.g. hydrophobic,hydrophilic, polar etc) could be exchanged for another amino acidresidue from the same functional group.

In some embodiments, such changes may be made in any position of thesequence of the IL-17A binding polypeptide as disclosed herein. In otherembodiments, such changes may be made only in the non-variablepositions, also denoted scaffold amino acid residues. In such cases,changes are not allowed in the variable positions, i.e. positionsdenoted with an “X” in sequence i).

The term “% identity”, as used throughout the specification, may forexample be calculated as follows. The query sequence is aligned to thetarget sequence using the CLUSTAL W algorithm (Thompson et al, NucleicAcids Research, 22: 4673-4680 (1994)). A comparison is made over thewindow corresponding to the shortest of the aligned sequences. Theshortest of the aligned sequences may in some instances be the targetsequence. In other instances, the query sequence may constitute theshortest of the aligned sequences. The amino acid residues at eachposition are compared and the percentage of positions in the querysequence that have identical correspondences in the target sequence isreported as % identity.

In one particular embodiment according to the first aspect, there isprovided a polypeptide as defined above, wherein, in sequence i),

X₂ is selected from A, H and M;

X₄ is selected from A, D, E, F, L, M, N, Q, R and Y;

X₁₁ is selected from A, D, E, F, G, L, M, N, S, T and Y;

X₁₈ is selected from A, D, E and V;

X₂₀ is selected from A, G, Q and W;

X₂₁ is selected from E, F, H, N, R, T, V, W and Y;

X₂₅ is selected from A, D, E, G, H, I, L, N, Q, R, S, T and V; and

X₂₈ is selected from I, N and R.

In another particular embodiment according to the first aspect, there isprovided a polypeptide as defined in the paragraph immediately above,wherein in addition, in sequence i),

X₁₆ is is T,

X₁₇ is W;

X₂₁ is selected from E, F, H, W, T and Y;

X₂₅ is selected from A, D, E, G, H, I, L, N, Q, R, S and T;

X₂₆ is K; and

X₂₉ is D.

“X_(n)” and “X_(m)” are used herein to indicate amino acids in positionsn and m in the sequence i) as defined above, wherein n and m areintegers indicating the position of an amino acid within sequence i) ascounted from the N terminus. For example, X₃ and X₇ indicate the aminoacids in positions three and seven, respectively, from the N-terminalend of sequence i).

In embodiments according to the first aspect, there are providedpolypeptides wherein X_(n) in sequence i) is independently selected froma group of possible residues as listed in Table 1. The skilled personwill appreciate that X_(n) may be selected from any one of the listedgroups of possible residues and that this selection is independent fromthe selection of amino acids in X_(m), wherein n≠m. Thus, any of thelisted possible residues in position X_(n) in Table 1 may beindependently combined with any of the listed possible residues anyother variable position in Table 1.

The skilled person will appreciate that Table 1 is to be read asfollows: In one embodiment according to the first aspect, there isprovided a polypeptide wherein amino acid residue “X_(n)” in sequence i)is selected from “Possible residues”. Thus, Table 1 discloses severalspecific and individualized embodiments of the first aspect of thepresent disclosure. For example, in one embodiment according to thefirst aspect, there is provided a polypeptide wherein X₄ in sequence i)is selected from A, D, E, F, L, N, Q, R and Y, and in another embodimentaccording to the first aspect, there is provided a polypeptide whereinX₄ in sequence i) is selected from D, E, N and Y. For avoidance ofdoubt, the listed embodiments may be freely combined in yet otherembodiments. For example, one such combined embodiment is a polypeptidein which X₄ is selected from D, E, F, N, Q, R and Y, while X₇ isselected from F, V and W, and X₁₈ is selected from A, D, E and H, and soon.

TABLE 1 X_(n) Possible residues X₂ A, M, Y X₂ A, M X₂ A X₄ A, D, E, F,K, L, M, N, Q, R, Y X₄ A, D, E, F, L, M, N, Q, R, S, Y X₄ A, D, E, F, L,M, N, Q, R, Y X₄ A, D, E, F, L, N, Q, R, Y X₄ A, D, E, F, M, N, Q, Y X₄A, D, E, F, L, Q, R X₄ A, D, E, L, Q, R X₄ A, D, Q, R X₄ D, Q X₄ A, D,E, F, N, Q, Y X₄ D, E, F, N, Q, R, Y X₄ D, E, N, Q, Y X₄ D, E, N, Y X₄D, E, Q, Y X₄ D, E, Y X₄ D, E X₄ D X₄ Q X₆ A, Q X₆ A, W X₆ Q, W X₆ W X₆A X₆ Q X₇ F, I, L, V, W, Y X₇ F, I, L, V, Y X₇ I, L, V, Y X₇ L, V, Y X₇L, V X₇ F, M, V, W, Y X₇ F, V, W, Y X₇ F, V, W X₇ F, V X₇ V X₇ L X₇ YX₁₀ A X₁₀ W X₁₁ A, D, E, F, G, L, M, N, S, T, Y X₁₁ A, D, E, F, L, M, S,T X₁₁ A, D, E, F, G, L, M, N, S, Y X₁₁ A, D, E, F, G, S, Y X₁₁ A, D, E,F, L, M, S X₁₁ A, D, E, L, M, S X₁₁ A, D, E, L, S X₁₁ A, D, E, L, M X₁₁A, D, E, L X₁₁ D, E, L X₁₁ D, L X₁₁ A, D, E, F, S, Y X₁₁ A, D, E, S X₁₁A, D, S X₁₁ A, S X₁₁ A, D X₁₁ D, S X₁₁ L X₁₁ D X₁₁ S X₁₁ A X₁₁ E X₁₆ TX₁₆ N X₁₇ H, W X₁₇ Y, W X₁₇ H, Y X₁₇ W X₁₇ H X₁₇ Y X₁₈ A, D, E, V X₁₈ A,D, E, H X₁₈ A, D, H X₁₈ A, D, E X₁₈ A, D X₁₈ D, E X₁₈ D X₁₈ A X₂₀ A, G,Q, W X₂₀ A, Q, S, W X₂₀ A, Q, W X₂₀ G, Q, W X₂₀ G, W X₂₀ Q, W X₂₀ A, WX₂₀ W X₂₀ A X₂₀ Q X₂₁ A, D, E, F, H, N, R, T, V, W, Y X₂₁ A, D, E, F, H,N, R, V, W, Y X₂₁ E, F, H, N, R, V, W, Y X₂₁ F, H, N, R, V, W, Y X₂₁ E,F, H, T, W, Y X₂₁ E, F, H, W, Y X₂₁ F, H, R, W, Y X₂₁ F, H, W, Y X₂₁ F,W, Y X₂₁ W, Y X₂₁ F, Y X₂₁ F, W X₂₁ Y X₂₁ W X₂₁ F X₂₅ A, D, E, G, H, I,L, N, Q, R, S, T, V X₂₅ A, D, E, G, H, I, L, N, Q, R, S, T X₂₅ A, D, E,G, H, N, Q, R, S, T, V X₂₅ D, E, G, N, Q, R, S, T, V X₂₅ D, E, N, Q, R,V X₂₅ A, D, E, G, I, L, N, Q, R, S, T X₂₅ D, E, G, N, Q, R, S X₂₅ D, E,N, Q, R, S X₂₅ A, E, G, L, N, Q, R, S, T X₂₅ E, N, Q, R, S X₂₅ E, N, Q,S, T X₂₅ E, N, Q, S X₂₅ N, Q, R X₂₅ E, Q, R X₂₅ Q, R, S X₂₅ Q, S X₂₅ Q,R X₂₅ Q X₂₅ S X₂₅ N X₂₅ E X₂₆ K X₂₆ S X₂₈ I, N, R X₂₈ I, L, R X₂₈ I, RX₂₈ N, R X₂₈ I, N X₂₈ R X₂₈ I X₂₈ N X₂₉ D X₂₉ R

In a more specific embodiment defining a sub-class of IL-17A bindingpolypeptides, sequence i) fulfills at least six of the eleven conditionsI-XI:

-   -   I. X₂ is A;    -   II. X₄ is selected from D, E and Q;    -   III. X₈ is A;    -   IV. X₇ is selected from F and V;    -   V. X₁₈ is T,    -   VI. X₁₇ is W;    -   VII. X₁₈ is selected from A and D;    -   VIII. X₂₀ is W;    -   IX. X₂₆ is K;    -   X. X₂₈ is IR; and    -   XI. X₂₉ is D.

In some examples of an IL-17A binding polypeptide according to the firstaspect, sequence i) fulfills at least seven of the eleven conditionsI-XI. More specifically, sequence i) may fulfill at least eight of theeleven conditions I-XI, such as at least nine of the eleven conditionsI-XI, such as at least ten of the eleven conditions I-XI, such as all ofthe eleven conditions I-XI.

In some embodiments of an IL-17A binding polypeptide according to thefirst aspect, X₂X₆, X₂X₁₀ or X₆X₁₀ is AA. In some embodiments, X₂X₁₇,X₂X₂₀, X₆X₁₇, X₆X₂₀, X₁₀X₁₇ or X₁₀X₂₈ is AW. In some embodiments, X₂X₂₈,X₆X₂₈ or X₁₀X₂₈ is AR. In some embodiments, X₁₇X₂₈ or X₂₀X₂₈ is WR. Insome embodiments, X₁₇X₂₀ is WW.

As described in detail in the experimental section to follow, theselection of IL-17A binding polypeptide variants has led to theidentification of a number of individual IL-17A binding motif (BM)sequences. These sequences constitute individual embodiments of sequencei) according to this aspect. The sequences of individual IL-17A bindingmotifs correspond to amino acid positions 8-36 in SEQ ID NO:1-1216presented in FIG. 1. Hence, in one embodiment of the IL-17A bindingpolypeptide according to this aspect, sequence i) corresponds to thesequence from position 8 to position 36 in a sequence selected from thegroup consisting of SEQ ID NO:1-1216. In one embodiment, sequence i)corresponds to the sequence from position 8 to position 36 in a sequenceselected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and1214. In one embodiment, sequence i) corresponds to the sequence fromposition 8 to position 36 in a sequence selected from the groupconsisting of SEQ ID NO:1-66. In one embodiment, sequence i) correspondsto the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-35. In one embodiment, sequencei) corresponds to the sequence from position 8 to position 36 in asequence selected from the group consisting of SEQ ID NO:1-27. In oneembodiment, sequence i) corresponds to the sequence from position 8 toposition 36 in a sequence selected from the group consisting of SEQ IDNO:1-10. In one embodiment, sequence i) corresponds to the sequence fromposition 8 to position 36 in a sequence selected from the groupconsisting of SEQ ID NO:1-7. In one embodiment, sequence i) correspondsto the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-4. In one embodiment, sequencei) corresponds to the sequence from position 8 to position 36 in SEQ IDNO:1.

In some embodiments of the present disclosure, the BM as defined above“forms part of” a three-helix bundle protein domain. This is understoodto mean that the sequence of the BM is “inserted” into or “grafted” ontothe sequence of the original three-helix bundle domain, such that the BMreplaces a similar structural motif in the original domain. For example,without wishing to be bound by theory, the BM is thought to constitutetwo of the three helices of a three-helix bundle, and can thereforereplace such a two-helix motif within any three-helix bundle. As theskilled person will realize, the replacement of two helices of thethree-helix bundle domain by the two BM helices has to be performed soas not to affect the basic structure of the polypeptide. That is, theoverall folding of the Ca backbone of the polypeptide according to thisembodiment of the invention is substantially the same as that of thethree-helix bundle protein domain of which it forms a part, e.g. havingthe same elements of secondary structure in the same order etc. Thus, aBM according to the disclosure “forms part” of a three-helix bundledomain if the polypeptide according to this embodiment of the aspect hasthe same fold as the original domain, implying that the basic structuralproperties are shared, those properties e.g. resulting in similar CDspectra. The skilled person is aware of other parameters that arerelevant.

In particular embodiments, the IL-17A binding motif (BM) thus forms partof a three-helix bundle protein domain. For example, the BM mayessentially constitute two alpha helices with an interconnecting loop,within said three-helix bundle protein domain. In particularembodiments, said three-helix bundle protein domain is selected fromdomains of bacterial receptor proteins. Non-limiting examples of suchdomains are the five different three-helical domains of Protein A fromStaphylococcus aureus, such as domain B, and derivatives thereof. Insome embodiments, the three-helical bundle protein domain is a variantof protein Z, which is derived from domain B of staphylococcal ProteinA.

In some embodiments where the IL-17A binding polypeptide as disclosedherein forms part of a three-helix bundle protein domain, the IL-17Abinding polypeptide may comprise an amino acid sequence binding module(BMod) selected from:

(SEQ ID NO: 1296) K-[BM]-DPSQSX_(a)X_(b)LLX_(c)EAKKLX_(d)X_(e)X_(f)Q,

wherein

-   -   [BM] is an IL-17A binding motif as defined herein, provided that        X₂₉ is

X_(a) is selected from A and S;

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S;

X_(e) is selected from D, E and S;

X_(f) is selected from A and S; and

-   iv) an amino acid sequence which has at least 85% identity to a    sequence defined by iii).

It may be beneficial in some embodiments that said polypeptides exhibithigh structural stability, such as resistance to chemical modifications,changes in physical conditions and proteolysis, during production orstorage, as well as in vivo. Thus, in other embodiments where the IL-17Abinding polypeptide as disclosed herein forms part of a three-helixbundle protein domain, the IL-17A binding polypeptide may comprise anamino acid sequence binding module (BMod) selected from:

(SEQ ID NO: 1297) K-[BM]-QPEQSX_(a)X_(b)LLX_(c)EAKKLX_(d)X_(e)X_(f)Q,

wherein

-   -   [BM] is an IL-17A binding motif as defined herein, provided that        X₂₉ is R;

X_(a) is selected from A and S;

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S;

X_(e) is selected from D, E and S;

X_(f) is selected from A and S; and

-   vi) an amino acid sequence which has at least 85% identity to a    sequence defined by v).

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences, which changes do not largely affect thetertiary structure or function of the polypeptide, are also within thescope of the present disclosure. Thus, in some embodiments, sequence ivand vi) have at least at least 87%, such as at least 89%, such as atleast 91%, such as at least 93%, such as at least 95%, such as at least97% identity to a sequence defined by iii) or v), respectively.

In one embodiment, X_(a) in sequence iii) or v) is A.

In one embodiment, X_(a) in sequence iii) or v) is S.

In one embodiment, X_(b) in sequence iii) or v) is N.

In one embodiment, X_(b) in sequence iii) or v) is E.

In one embodiment, X_(c) in sequence iii) or v) is A.

In one embodiment, X_(c) in sequence iii) or v) is S.

In one embodiment, X_(c) in sequence iii) or v) is C.

In one embodiment, X_(d) in sequence iii) or v) is E.

In one embodiment, X_(d) in sequence iii) or v) is N.

In one embodiment, X_(d) in sequence iii) or v) is S.

In one embodiment, X_(e) in sequence iii) or v) is D.

In one embodiment, X_(e) in sequence iii) or v) is E.

In one embodiment, X_(e) in sequence iii) or v) is S.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is selected fromEE, ES, SD, SE and SS.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is ES.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is SE.

In one embodiment, X_(d)X_(e) in sequence iii) or v) is SD.

In one embodiment, X_(f) in sequence iii) or v) is A.

In one embodiment, X_(f) in sequence iii) or v) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is S and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A; X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is S, X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is S, X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is C; X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is A; X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is S, X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is A; X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is C; X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A; X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is S, X_(d)X_(e) is ES and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is C; X_(d)X_(e) is ES and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is A; X_(d)X_(e) is SD and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A; X_(d)X_(e) is SD and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is A; X_(b) is N; X_(c)is C; X_(d)X_(e) is SD and X_(f) is A.

In one embodiment, in sequence iii) or v), X_(a) is S; X_(b) is E; X_(c)is S, X_(d)X_(e) is SD and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is A; X_(d)X_(e) is SD and X_(f) is S.

In one embodiment, in sequence iii) or v), X_(a) is S, X_(b) is E; X_(c)is C; X_(d)X_(e) is SD and X_(f) is S.

In yet a further embodiment, sequence iii) corresponds to the sequencefrom position 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1-1216. In one embodiment, sequence iii)corresponds to the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and1214. In one embodiment, sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1-66. In one embodiment, sequence iii)corresponds to the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-35. In anotherembodiment, sequence iii) corresponds to the sequence from position 7 toposition 55 in a sequence selected from the group consisting of SEQ IDNO:1-27. In one embodiment, sequence iii) corresponds to the sequencefrom position 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1-10. In yet another embodiment, sequence iii)corresponds to the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-7. In one embodiment,sequence iii) corresponds to the sequence from position 7 to position 55in a sequence selected from the group consisting of SEQ ID NO:1-4 and inanother embodiment, sequence iii) corresponds to the sequence fromposition 7 to position 55 in SEQ ID NO:1.

Also, in a further embodiment, there is provided an IL-17A bindingpolypeptide, which comprises an amino acid sequence selected from:

vii) YA-[BMod]-AP (SEQ ID NO:1298),wherein [BMod] is an IL-17A binding module as defined above; andviii) an amino acid sequence which has at least 86% identity to asequence defined by vii).

In an alternative further embodiment, there is provided an IL-17Abinding polypeptide, which comprises an amino acid sequence selectedfrom:

ix) FA-[BMod]-AP (SEQ ID NO:1299),

wherein [BMod] is an IL-17A binding module as defined above; andx) an amino acid sequence which has at least 86% identity to a sequencedefined by ix).

Alternatively, there is provided an IL-17A binding polypeptide, whichcomprises an amino acid sequence selected from:

xi) FN-[BMod]-AP (SEQ ID NO:1300),

wherein [BMod] is an IL-17A binding module as defined above; andxii) an amino acid sequence which has at least 86% identity to asequence defined by xi).

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences without largely affecting the tertiarystructure and the function thereof also fall within the scope of thepresent disclosure. Thus, in some embodiments, the IL-17A bindingpolypeptides as defined above may for example have a sequence which isat least 88%, such as at least 90%, such as at least 92%, such as atleast 94%, such as at least 96%, such as at least 98% identical to asequence defined by vii), ix) or xi).

In some embodiments, the IL-17A binding motif may form part of apolypeptide comprising an amino acid sequence selected from

SEQ ID NO:  1250 ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK, 1251ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK, 1252ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK, 1253ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK, 1254AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK, 1255VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK, 1256AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK, 1257VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK, 1258VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK, 1259AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1260AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP, 1261AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAPK, 1262AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAP, 1263AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1264AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP, 1265AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK, 1266AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1267AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP, 1268AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1269AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP, 1270AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK, 1271AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK, 1272AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK, 1273AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP, 1274AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK, 1275AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK, 1276AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK, 1277AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP, 1278AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK, 1279AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK, 1280AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK, 1281VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1282VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1283VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK, 1284VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK, 1285VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK, 1286VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK, 1287VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK, 1288VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK, 1289VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK, 1290VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK, 1291VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK, 1292VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK, 1293AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK, and 1294ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,wherein [BM] is an IL-17A binding motif as defined above.

In one embodiment, the IL-17A binding polypeptide comprises an aminoacid sequence selected from:

(SEQ ID NO: 1281) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

wherein [BM] is an IL-17A binding motif as defined above; and

-   xiv) an amino acid sequence which has at least 86% identity to the    sequence defined in xiii).

Again, polypeptides comprising minor changes as compared to the aboveamino acid sequences without largely affecting the tertiary structureand the function thereof are also within the scope of the presentdisclosure. Thus, in some embodiments, the IL-17A binding polypeptidesas defined above may for example have a sequence which is at least 87%,such as at least 89%, such as at least 91%, such as at least 93%, suchas at least 94%, such as at least 96%, such as at least 98% identical tothe sequence defined by xiii).

Sequence xiii) in such a polypeptide may be selected from the groupconsisting of SEQ ID NO:1-1216. In one embodiment, sequence xiii) isselected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and1214. In one embodiment, sequence xiii) is selected from the groupconsisting of SEQ ID NO:1-66. In one embodiment, sequence xiii) isselected from the group consisting of SEQ ID NO:1-35. In anotherembodiment, sequence xiii) is selected from the group consisting of SEQID NO:1-27. In one embodiment, sequence xiii) is selected from the groupconsisting of SEQ ID NO:1-10. In one embodiment, sequence xiii) isselected from SEQ ID NO:1-7. In one embodiment, sequence xiii) isselected from the group consisting of SEQ ID NO:1-4. In one embodiment,sequence xiii) is SEQ ID NO:1.

In one embodiment, the IL-17A binding polypeptide comprises an aminoacid sequence selected from:

(SEQ ID NO: 1259) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

wherein [BM] is an IL-17A binding motif as defined above; and

-   xvi) an amino acid sequence which has at least 86% identity to the    sequence defined in xv).

Again, polypeptides comprising minor changes as compared to the aboveamino acid sequences without largely affecting the tertiary structureand the function thereof are also within the scope of the presentdisclosure. Thus, in some embodiments, the IL-17A binding polypeptidesas defined above may for example have a sequence which is at least 87%,such as at least 89%, such as at least 91%, such as at least 93%, suchas at least 94%, such as at least 96%, such as at least 98% identical tothe sequence defined by xv).

Sequence xv) in such a polypeptide may be selected from the groupconsisting of SEQ ID NO:1217-1222. In one embodiment, sequence xv) isselected from the group consisting of SEQ ID NO:1218-1222. In oneembodiment, sequence xv) is selected from the group consisting of SEQ IDNO:1219-1222. In another embodiment, sequence xv) is selected from thegroup consisting of SEQ ID NO:1219 and SEQ ID NO:1222. In oneembodiment, sequence xv) is SEQ ID NO:1219.

The small size and robustness of the IL-17A binding domains of thepresent disclosure confer several advantages over conventionalmonoclonal antibody based therapies. Such advantages include thepossibility of subcutaneous (s.c.) administration at higher doses thanantibodies, alternative routes of administration, flexibility informatting for superior potency and absence of Fc-mediated side effects.The small size combined with potential for very high solubility (>100mg/ml) and stability allows for extreme molar amounts of drug in a smallvolume for s.c. injections. For systemic administration, this suggestsoutpatient “home use” treatment using convenient small prefilledsyringes or auto-injectors, with low volume and well toleratedadministration of doses. In addition, the capacity for high molarconcentrations in drug preparations in combination with the ability toretain functional stability in diverse formulations opens up for topical(skin, eye, lung) administration routes. Psoriasis, asthma, uveitis anddry eye syndrome are examples of indications where alternativeadministration routes could be especially relevant in IL-17A mediateddisease.

The terms “IL-17A binding” and “binding affinity for IL-17A” as used inthis specification refer to a property of a polypeptide which may betested for example by ELISA, by use of surface plasmon resonance (SPR)technology, or by use of the Kinetic Exclusion Assay (KinExA®). Forexample as described in the examples below, IL-17A binding affinity maybe tested in an experiment in which samples of the polypeptide arecaptured on antibody coated ELISA plates and biotinylated IL-17A,followed by streptavidin conjugated HRP, are added. TMB substrate isadded and the absorbance at 450 nm is measured using a multi-well platereader, such as Victor³ (Perkin Elmer). The skilled person may theninterpret the results obtained by such experiments to establish at leasta qualitative measure of the binding affinity of the polypeptide forIL-17A. If a quantitative measure is desired, for example to determinethe EC50 value (the half maximal effective concentration) for theinteraction, ELISA may also be used. The response of the polypeptidesagainst a dilution series of biotinylated IL-17A are measured usingELISA as described above. The skilled person may then interpret theresults obtained by such experiments and EC50 values may be calculatedfrom the results using for example GraphPad Prism 5 and non-linearregression.

IL-17A binding affinity may also be tested in an experiment in whichIL-17A, or a fragment thereof, is immobilized on a sensor chip of thesurface plasmon resonance (SPR) instrument, and the sample containingthe polypeptide to be tested is passed over the chip. Alternatively, thepolypeptide to be tested is immobilized on a sensor chip of theinstrument, and a sample containing IL-17A, or a fragment thereof, ispassed over the chip. The skilled person may then interpret the resultsobtained by such experiments to establish at least a qualitative measureof the binding affinity of the polypeptide for IL-17A. If a quantitativemeasure is desired, for example to determine a K_(D) value for theinteraction, surface plasmon resonance methods may also be used. Bindingvalues may for example be defined in a Biacore (GE Healthcare) orProteOn XPR 36 (Bio-Rad) instrument. IL-17A is suitably immobilized on asensor chip of the instrument, and samples of the polypeptide whoseaffinity is to be determined are prepared by serial dilution andinjected in random order. K_(D) values may then be calculated from theresults using for example the 1:1 Langmuir binding model of theBIAevaluation 4.1 software, or other suitable software, provided by theinstrument manufacturer.

Another method for determining binding affinity for IL-17A is theKinetic Exclusion Assay (KinExA®; Sapidyne Instruments Inc, Boise, USA;Darling and Brault, 2004. Assay and Drug Dev Tech 2(6):647-657) formeasurements of the equilibrium binding affinity and kinetics betweenunmodified molecules in solution. For affinity analysis, the equilibriumdissociation constant, K_(D), and the rate of association, k_(a), areexperimentally determined, while the rate of dissociation, k_(d), may becalculated based on the equation k_(d)=K_(D)*k_(a).

A KinExA® K_(D) analysis requires immobilization of one interactionpartner (e.g. the titrated binding partner) to a solid phase, which isthen used as a probe to capture the other interaction partner (e.g. theconstant binding partner) free in solution once an equilibrium isreached. For each experiment, a series of solutions with a constantconcentration of one binding partner and a titration of the otherbinding partner are equilibrated. The solutions are then briefly exposedto the solid phase and a portion of free constant binding partner iscaptured and labeled with a fluorescent secondary molecule. The shortcontact time with the solid phase is less than the time needed fordissociation of the pre-formed complex in solution, meaning thatcompetition between the solution and the solid phase titrated bindingpartner is “kinetically excluded”. Since the solid phase is only used asa probe for the free constant binding partner in each sample, thesolution equilibrium is not altered during measurements. A K_(D) valueis calculated from signals generated from captured free constant bindingpartner, which are directly proportional to the concentration of freeconstant binding partner in the equilibrated sample. The data may beanalyzed using the KinExA® Pro software and least squares analysis tofit the optimal solutions for the K_(D) and the Active Binding siteConcentration (ABC) to a curve representative of a stoichiometricrelevant model, for instance a 1:1 reversible bi-molecular interaction.

Determination of binding kinetics may be done in a similar format as theequilibrium analysis, except measurements are collected“pre-equilibrium” and the binding signals are a function of time andtotal concentration of the titrated binding partner. There are twomethods that can be used to determine the k_(a). The “direct method”holds the concentrations of titrated and constant binding partnersfixed, and the solution is probed over time. The amount of the freeconstant binding partner in the solution will decrease as the samplemoves toward equilibrium. The “inject method” holds incubation time andone partner's concentration fixed, while titrating concentrations of theother partner. As the concentration of the titrated binding partnerincreases, the amount of free constant binding partner will decrease asmore complexes are formed.

In one embodiment, the IL-17A binding polypeptide is capable of bindingto IL-17A such that the K_(D) value of the interaction is at most 1×10⁻⁶M, such as at most 1×10⁻⁷ M, such as at most 1×10⁻⁸ M, such as at most1×10⁻⁹ M.

In one embodiment, an IL-17A binding polypeptide according to any aspectdisclosed herein is capable of binding to an IL-17A molecule selectedfrom the group consisting of human IL-17A and murine IL-17A. In oneembodiment, the IL-17A binding polypeptide is capable of binding tohuman IL-17A. In one embodiment, the IL-17A binding polypeptide iscapable of binding to murine IL-17A. In one embodiment, the IL-17Abinding polypeptide is capable of binding to human IL-17A and to murineIL-17A. In this regard, human IL-17A may comprise the amino acidsequence SEQ ID NO:1226, or an antigenically effective fragment thereof.Likewise, murine IL-17A may comprise the amino acid sequence SEQ IDNO:1227, or an antigenically effective fragment thereof.

The skilled person will understand that various modifications and/oradditions can be made to an IL-17A binding polypeptide according to anyaspect disclosed herein in order to tailor the polypeptide to a specificapplication without departing from the scope of the present disclosure.

For example, in one embodiment, there is provided an IL-17A bindingpolypeptide as described herein, which polypeptide has been extended byand/or comprises additional amino acids at the C terminus and/or Nterminus. Such a polypeptide should be understood as a polypeptidehaving one or more additional amino acid residues at the very firstand/or the very last position in the polypeptide chain, i.e. at the N-and/or C-terminus of sequence i) or ii). Thus, an IL-17A bindingpolypeptide may comprise any suitable number of additional amino acidresidues, for example at least one additional amino acid residue. Eachadditional amino acid residue may individually or collectively be addedin order to, for example, improve and/or simplify production,purification, stabilization in vivo or in vitro, coupling or detectionof the polypeptide. Such additional amino acid residues may comprise oneor more amino acid residues added for the purpose of chemical coupling.One example of this is the addition of a cysteine residue. Additionalamino acid residues may also provide a “tag” for purification ordetection of the polypeptide, such as a His₆ tag, a (HisGlu)₃ tag(“HEHEHE” tag) or a “myc” (c-myc) tag or a “FLAG” tag for interactionwith antibodies specific to the tag or immobilized metal affinitychromatography (I MAC) in the case of the His₆-tag.

The further amino acids as discussed above may be coupled to the IL-17Abinding polypeptide by means of chemical conjugation (using knownorganic chemistry methods) or by any other means, such as expression ofthe IL-17A binding polypeptide as a fusion protein or joined in anyother fashion, either directly or via a linker, for example an aminoacid linker.

The further amino acids as discussed above may for example comprise oneor more polypeptide domain(s). A further polypeptide domain may providethe IL-17A binding polypeptide with another function, such as forexample yet another binding function, an enzymatic function, a toxicfunction, a fluorescent signaling function or combinations thereof.

A further polypeptide domain may moreover provide another IL-17A bindingmoiety with the same IL-17A binding function. Thus, in a furtherembodiment, there is provided an IL-17A binding polypeptide in amultimeric form. Said multimer is understood to comprise at least twoIL-17A binding polypeptides as disclosed herein as monomer units, theamino acid sequences of which may be the same or different. Multimericforms of the polypeptides may comprise a suitable number of domains,each having an IL-17A binding motif, and each forming a monomer withinthe multimer. These domains may have the same amino acid sequence, butalternatively, they may have different amino acid sequences. In otherwords, the IL-17A binding polypeptide of the invention may form homo- orheteromultimers, for example homo- or heterodimers. In one embodiment,there is provided an IL-17A binding polypeptide, wherein said monomericunits are covalently coupled together. In another embodiment, saidIL-17A binding polypeptide monomer units are expressed as a fusionprotein. In one embodiment, there is provided an IL-17A bindingpolypeptide in dimeric form.

Additionally, “heterogenic” fusion polypeptides or proteins, orconjugates, in which an IL-17A binding polypeptide described herein, ormultimer thereof, constitutes a first domain, or first moiety, and thesecond and further moieties have other functions than binding IL-17A,are also contemplated and fall within the ambit of the presentdisclosure. The second and further moiety/moieties of the fusionpolypeptide or conjugate in such a protein suitably have a desiredbiological activity.

Thus, in a second aspect of the present disclosure, there is provided afusion protein or a conjugate, comprising a first moiety consisting ofan IL-17A binding polypeptide according to the first aspect, and asecond moiety consisting of a polypeptide having a desired biologicalactivity. In another embodiment, said fusion protein or conjugate mayadditionally comprise further moieties, comprising desired biologicalactivities that can be either the same or different from the biologicalactivity of the second moiety.

Non-limiting examples of a desired biological activity comprise atherapeutic activity, a binding activity and an enzymatic activity.

In one embodiment, the second moiety having a desired biologicalactivity is a therapeutically active polypeptide.

Non-limiting examples of therapeutically active polypeptides arebiomolecules, such as molecules selected from the group consisting ofhuman endogenous enzymes, hormones, growth factors, chemokines,cytokines and lymphokines. Non-limiting examples of contemplatedcytokines are IL-2, IL-4, IL-7, IL-10, IL-11, IL-13, IL-21, IL-27,IL-35, IFNβ and TGFβ. Non-limiting examples of contemplated chemokinesare SDF-1/CXCL12, BCL/BCA-1/CXCL13, CXCL16, HCC1/CCL14, TARC/CCL17,PARC/CCL18, MIP-3β/ELC/CCL19, SLC/CCL21, CCL25/TECK and CCL27/CTACK.

Non-limiting examples of binding activities are binding activities whichincrease the in vivo half-life of the fusion protein or conjugate. Inone particular embodiment, said binding activity is an albumin bindingactivity which increases the in vivo half-life of the fusion protein orconjugate. In one embodiment, said albumin binding activity is providedby the albumin binding domain of streptococcal protein G or a derivativethereof. In another particular embodiment, said binding activity is anFcRn binding activity which increases the in vivo half-life of thefusion protein or conjugate.

In one embodiment, the fusion protein or conjugate of this second aspectcomprises two monomers of the IL-17A binding polypeptide of the firstaspect, whose amino acid sequences may be the same or different, linkedby an albumin binding moiety. In a specific embodiment of thisconstruct, the fusion protein or conjugate comprises two IL-17A bindingmonomers with an albumin binding moiety between them. Said albuminbinding moiety may e.g. be a “GA” albumin binding domain fromstreptococcal protein G, such as “GA3”, or a derivative thereof asdescribed in any one of WO2009/016043, WO2012/004384, WO2014/048977 andWO2015/091957.

In one embodiment, the format of such a fusion protein or conjugate is“Z-A-Z”, where each “Z” individually is an IL-17A binding polypeptide asdescribed herein, and “A” is an albumin binding domain. In oneembodiment, such an IL-17A binding polypeptide with the “Z-A-Z” formatis capable of binding to IL-17A such that the K_(D) value of theinteraction is at most 1×10⁻¹⁰ M, such as at most 1×10⁻¹¹ M, such as atmost 1×10⁻¹² M, such as at most 1×10⁻¹³ M. In one embodiment, such a“Z-A-Z” polypeptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:1233-1247, for example selected from thegroup consisting of SEQ ID NO:1236, 1237 and 1242-1247, such as selectedfrom the group consisting of SEQ ID NO:1236, 1244 and 1247 or the groupconsisting of SEQ ID NO:1237, 1244 and 1247. In an even more specificembodiment, the “Z-A-Z” polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1244 and 1245. In oneembodiment, the “Z-A-Z” polypeptide comprises SEQ ID NO:1244.

In one embodiment, said binding activity is binding to an angiogenesisassociated factor. Non-limiting examples of angiogenesis associatedfactors include fibroblast growth factor (FGF), fibroblast growth factor1 (FGF-1), basic FGF, angiogenin 1 (Ang-1), angiogenin 2 (Ang-2),angiopoietin 1 (Angpt-1), angiopoietin 2 (Angpt-2), angiopoietin 3(Angpt-3), angiopoietin 4 (Angpt-4), tyrosine kinase withimmunoglobulin-like domains 1 (TIE-1), tyrosine kinase withimmunoglobulin-like domains 2 (TIE-2), vascular endothelial growthfactor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor2 (VEGFR-2), vascular endothelial growth factor receptor 3 (VEGFR-3),vascular endothelial growth factor A (VEGF-A), vascular endothelialgrowth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C),vascular endothelial growth factor D (VEGF-D), vascular endothelialgrowth factor E (VEGF-E), placental growth factor (PIGF), transforminggrowth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2),transforming growth factor β receptors (type I, type II and type III),matrix metalloproteinase (MMP), MET receptor tyrosine kinase (alsodenoted cMET and hepatocyte growth factor receptor (HGFR)), members ofthe Notch family of receptors and beta-catenin.

In one embodiment, said binding activity is binding to an immuneresponse associated factor. Non-limiting examples of immune responseassociated factors include

-   -   T-cell regulatory factors such as CD3, CD4, CD6, CD28, T-cell        receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic        T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell        death protein 1 (PD-1), programmed death-ligand 1 (PD-L1),        programmed death-ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7        homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and        T-lymphocyte attenuator (BTLA), killer inhibitory receptor        (KIR), lymphocyte-activation gene 3 (LAG3), galectin-9 (Gal9)/T        cell immunoglobulin mucin-3 (TIM3) and adenosine/alpha-2        adrenergic receptors (A2aR);    -   NK-cell recruitment factors such as CD16, natural killer cell        lectin-like receptor gene 2D product (NKG2D), lymphocyte        function-associated antigen 1 (LFA1) and the natural        cytotoxicity receptors NKp30 and NKp40;    -   inflammation-associated factors such as        -   cytokines and their receptors, including tumor necrosis            factors (TNF); tumor necrosis factor ligand super family            (TNFSF) members TNFSF11/RANKL, TNFSF12/TWEAK, TNFSF13,            TNFSF13B/BAFF/BLys, TNFSF14, TNFSF15; interleukins (IL)            IL-1α, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-12, IL-17B,            IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-22, IL-23, IL-26,            IL-32, IL33 and IL-34; interferons INFα and INFγ;            granulocyte colony-stimulating factor (GCSF), granulocyte            colony-stimulating factor (GM-CSF); and        -   inflammatory chemokines and their receptors, including            IL-8/CXCL8, ENA-78/CXCL5, GROa/CXCL1, CTAP-III/CXCL7,            IP-10/CXCL10, Mig/CXCL9, PF4/CXCL4, GCP-2/CXCL6, MCP-1/CCL2,            MIP-1α/CCL3, MIP-3α/CCL20, RANTES/CCL5; lymphotactin/XCL1            and fractalkine/CX3CL1.

In particularly selected embodiments, the binding activity of saidsecond moiety is binding to a target selected from the group consistingof TNF, IL-1β, IL-6, IL-17F and IL-23.

In one embodiment of either the first or second aspect of the presentdisclosure, there is provided an IL-17A binding polypeptide, fusionprotein or conjugate which comprises an immune response modifying agent.Non-limiting examples of such immune response modifying agents includeimmunosuppressive or immunomodulating agents or other anti-inflammatoryagents. For example, an IL-17A binding polypeptide, fusion protein orconjugate as described herein may comprise an agent selected from thegroup consisting of disease-modifying antirheumatic drugs (DMARDs), suchas gold salts, azathioprine, methotrexate and leflunomide; calcineurininhibitors, such as cyclosporin A or FK 506; modulators of lymphocyterecirculation; mTOR inhibitors, such as rapamycin; an ascomycin havingimmunosuppressive properties; glucocorticoids; corticosteroids;cyclophosphamide; immunosuppressive monoclonal antibodies, for examplemonoclonal antibodies with affinity for leukocyte receptors such as MHC,CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 ortheir ligands; adhesion molecule inhibitors, such as LFA-1 antagonists,ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists;anti-TNF agents, such as etanercept and monoclonal antibodies to TNFsuch as infliximab and adalimumab; inhibitors of proinflammatorycytokines; IL-1 blockers such as anakinra or IL-1 trap; IL-6 blockers;chemokine inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs)such as aspirin; and anti-infectious agents and other immune responsemodulating agents; as well as combinations of any two or more of theabove.

In one embodiment of either the first or second aspect of the presentdisclosure, there is provided an IL-17A binding polypeptide, fusionprotein or conjugate which comprises a toxic compound. Non-limitingexamples of such toxic compounds include calicheamicin, maytansinoid,neocarzinostatin, esperamicin, dynemicin, kedarcidin, maduropeptin,doxorubicin, daunorubicin, auristatin, ricin-A chain, modeccin,truncated Pseudomonas exotoxin A, diphtheria toxin and recombinantgelonin.

Recently, considerable progress has been made in the development ofmultispecific agents, such as antibodies with the ability to bind tomore than one antigen, for example through engineering of thecomplementarity determining regions (CDRs) to address two antigens in asingle antibody combining site (Bostrom et al, 2009, Science323(5921):1610-1614, Schaefer et al, 2011, Cancer Cell 20(4):472-486),via construction of heterodimeric antibodies using engineered Fc units(Carter, 2001, J Immunol Methods 248(1-2):7-15, Schaefer et al, 2011,Proc Natl Acad Sci USA 108(27):11187-11192) and via genetic fusion ofauxiliary recognition units to N- or C-termini of light or heavy chainsof full-length antibodies (Kanakaraj et al, 2012, MAbs 4(5):600-613,LaFleur et al, 2013, MAbs 5(2):208-218).

As discussed above, it may be beneficial for a polypeptide with affinityfor IL-17A as disclosed herein to also exhibit affinity for anotherfactor, such as an immune response associated factor, for example aninflammation-associated factor.

Thus, in a third aspect of the present disclosure, there is provided acomplex comprising at least one IL-17A binding polypeptide as definedherein and at least one antibody or an antigen binding fragment thereof.

When used herein to denote the third aspect of the disclosure, the term“complex” is intended to refer to two or more associated polypeptidechains, one having an affinity for IL-17A by virtue of its IL-17Abinding motif as defined above, and the other being an antibody or anantigen binding fragment thereof. These polypeptide chains may eachcontain different protein domains, as described amply above for theIL-17A binding polypeptide of the first and second aspects, and theresulting multiprotein complex can have multiple functions. “Complex”intends to refer to two or more polypeptides as defined herein,connected by covalent bonds, for example two or more polypeptide chainsconnected by covalent bonds through expression thereof as a recombinantfusion protein, or associated by chemical conjugation.

The third aspect provides a complex comprising an antibody or an antigenbinding fragment thereof. As is well known, antibodies areimmunoglobulin molecules capable of specific binding to a target (anantigen), such as a carbohydrate, polynucleotide, lipid, polypeptide orother, through at least one antigen recognition site located in thevariable region of the immunoglobulin molecule. As used herein, the term“antibody or an antigen binding fragment thereof” encompasses not onlyfull-length or intact polyclonal or monoclonal antibodies, but alsoantigen-binding fragments thereof, such as Fab, Fab′, F(ab′)₂, Fab₃, Fvand variants thereof, fusion proteins comprising one or more antibodyportions, humanized antibodies, chimeric antibodies, minibodies,diabodies, triabodies, tetrabodies, linear antibodies, single chainantibodies, multispecific antibodies (e.g. bispecific antibodies) andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies and covalently modified antibodies. Furtherexamples of modified antibodies and antigen binding fragments thereofinclude nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs(bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dualacting Fab), two-in-one antibodies, SMIPs (small modularimmunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs(dual variable domain immunoglobulin), CovX-bodies (peptide modifiedantibodies), duobodies and triomAbs. This listing of variants ofantibodies and antigen binding fragments thereof is not to be seen aslimiting, and the skilled person is aware of other suitable variants.

A full-length antibody comprises two heavy chains and two light chains.Each heavy chain contains a heavy chain variable region (V_(H)) andfirst, second and third constant regions (C_(H)1, C_(H)2 and CH3). Eachlight chain contains a light chain variable region (V_(L)) and a lightchain constant region (C_(L)). Depending on the amino acid sequence ofthe constant domain of its heavy chains, antibodies are assigned todifferent classes. There are six major classes of antibodies: IgA, IgD,IgE, IgG, IgM and IgY, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theterm “full-length antibody” as used herein refers to an antibody of anyclass, such as IgD, IgE, IgG, IgA, IgM or IgY (or any sub-classthereof). The subunit structures and three-dimensional configurations ofdifferent classes of antibodies are well known.

An “antigen binding fragment” is a portion or region of an antibodymolecule, or a derivative thereof, that retains all or a significantpart of the antigen binding of the corresponding full-length antibody.An antigen binding fragment may comprise the heavy chain variable region(V_(H)), the light chain variable region (V_(L)), or both. Each of theV_(H) and V_(L) typically contains three complementarity determiningregions CDR1, CDR2 and CDR3. The three CDRs in V_(H) or V_(L) areflanked by framework regions (FR1, FR2, FR3 and FR4). As briefly listedabove, examples of antigen binding fragments include, but are notlimited to: (1) a Fab fragment, which is a monovalent fragment having aV_(L)—C_(L) chain and a V_(H)-C_(H)1 chain; (2) a Fab′ fragment, whichis a Fab fragment with the heavy chain hinge region, (3) a F(ab′)₂fragment, which is a dimer of Fab′ fragments joined by the heavy chainhinge region, for example linked by a disulfide bridge at the hingeregion; (4) an Fc fragment; (5) an Fv fragment, which is the minimumantibody fragment having the V_(L) and V_(H) domains of a single arm ofan antibody; (6) a single chain Fv (scFv) fragment, which is a singlepolypeptide chain in which the V_(H) and V_(L) domains of an scFv arelinked by a peptide linker; (7) an (scFv)₂, which comprises two V_(H)domains and two V_(L) domains, which are associated through the twoV_(H) domains via disulfide bridges and (8) domain antibodies, which canbe antibody single variable domain (V_(H) or V_(L)) polypeptides thatspecifically bind antigens.

Antigen binding fragments can be prepared via routine methods. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of afull-length antibody molecule, and Fab fragments can be generated byreducing the disulfide bridges of F(ab′)₂ fragments. Alternatively,fragments can be prepared via recombinant technology by expressing theheavy and light chain fragments in suitable host cells (e.g., E. coli,yeast, mammalian, plant or insect cells) and having them assembled toform the desired antigen-binding fragments either in vivo or in vitro. Asingle-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region. Forexample, a flexible linker may be incorporated between the two variableregions. The skilled person is aware of methods for the preparation ofboth full-length antibodies and antigen binding fragments thereof.

Thus, in one embodiment, this aspect of the disclosure provides acomplex as defined herein, wherein said at least one antibody or antigenbinding fragment thereof is selected from the group consisting offull-length antibodies, Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fc fragments, Fv fragments, single chain Fv fragments,(scFv)₂ and domain antibodies. In one embodiment, said at least oneantibody or antigen binding fragment thereof is selected fromfull-length antibodies, Fab fragments and scFv fragments. In oneparticular embodiment, said at least one antibody or antigen bindingfragment thereof is a full-length antibody.

In one embodiment of said complex as defined herein, the antibody orantigen binding fragment thereof is selected from the group consistingof monoclonal antibodies, human antibodies, humanized antibodies,chimeric antibodies, and antigen-binding fragments thereof.

The term “monoclonal antibodies” as used herein refers to antibodieshaving monovalent affinity, meaning that each antibody molecule in asample of the monoclonal antibody binds to the same epitope on theantigen, whereas the term “polyclonal antibodies” as used herein refersto a collection of antibodies that react against a specific antigen, butin which collection there may be different antibody molecules forexample identifying different epitopes on the antigen. Polyclonalantibodies are typically produced by inoculation of a suitable mammaland are purified from the mammal's serum. Monoclonal antibodies are madeby identical immune cells that are clones of a unique parent cell (forexample a hybridoma cell line). The term “human antibody” as used hereinrefers to antibodies having variable and constant regions correspondingsubstantially to, or derived from, antibodies obtained from humansubjects. The term “chimeric antibodies” as used herein, refers torecombinant or genetically engineered antibodies, such as for examplemouse monoclonal antibodies, which contain polypeptides or domains froma different species, for example human, introduced to reduce theantibodies' immunogenicity. The term “humanized antibodies” refers toantibodies from non-human species whose protein sequences have beenmodified to increase their similarity to antibody variants producednaturally in humans, in order to reduce immunogenicity.

It may be beneficial for a complex as defined herein to, in addition tobeing capable of binding IL-17A, target at least one additional antigen,such as an antigen selected from the group consisting of an antigenassociated with an angiogenesis related disorder and an antigenassociated with the immune response. In one embodiment, said additionalantigen is associated with angiogenesis. In one embodiment, saidadditional antigen is associated with the immune response.

In one embodiment, the antigen is associated with angiogenesis and isselected from the group consisting of fibroblast growth factor (FGF),fibroblast growth factor 1 (FGF-1), basic FGF, angiogenin 1 (Ang-1),angiogenin 2 (Ang-2), angiopoietin 1 (Angpt-1), angiopoietin 2(Angpt-2), angiopoietin 3 (Angpt-3), angiopoietin 4 (Angpt-4), tyrosinekinase with immunoglobulin-like domains 1 (TIE-1), tyrosine kinase withimmunoglobulin-like domains 2 (TIE-2), vascular endothelial growthfactor receptor 1 (VEGFR-1), vascular endothelial growth factor receptor2 (VEGFR-2), vascular endothelial growth factor receptor 3 (VEGFR-3),vascular endothelial growth factor A (VEGF-A), vascular endothelialgrowth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C),vascular endothelial growth factor D (VEGF-D), vascular endothelialgrowth factor E (VEGF-E), placental growth factor (PIGF), transforminggrowth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2),transforming growth factor β receptors (type I, type II and type III),matrix metalloproteinase (MMP), MET receptor tyrosine kinase (alsodenoted cMET and hepatocyte growth factor receptor (HGFR)), members ofthe Notch family of receptors and beta-catenin. In one embodiment, saidantibody or fragment thereof is selected from the group consisting ofAMG 780, AMG 386, MEDI-3617, nesvacumab, CVX-241, bevacizumab,ranibizumab, VGX100, CVX-241, ABP 215, PF-06439535, fresolimumab,metelimumab, onartuzumab, emibetuzumab and tarextumab.

In one embodiment, the antigen is associated with the immune response ora disorder of the immune system, and is selected from the groupconsisting of

-   -   T-cell regulatory factors such as CD3, CD4, CD6, CD28, T-cell        receptor α (TCRα), T-cell receptor β (TCRβ), cytotoxic        T-lymphocyte-associated protein 4 (CTLA-4), and programmed cell        death protein 1 (PD-1), programmed death-ligand 1 (PD-L1),        programmed death-ligand 2 (PD-L2), B7 homolog 3 (B7-H3), B7        homolog 4 (B7-H4), herpes virus entry mediator (HVEM)/B- and        T-lymphocyte attenuator (BTLA), killer inhibitory receptor        (KIR), lymphocyte-activation gene 3 (LAG3), galectin-9 (Gal9)/T        cell immunoglobulin mucin-3 (TIM3) and adenosine/alpha-2        adrenergic receptors (A2aR);    -   NK-cell recruitment factors such as CD16, natural killer cell        lectin-like receptor gene 2D product (NKG2D), lymphocyte        function-associated antigen 1 (LFA1) and the natural        cytotoxicity receptors NKp30 and NKp40;    -   inflammation-associated factors such as        -   cytokines and their receptors, including tumor necrosis            factors (TNF); tumor necrosis factor ligand super family            (TNFSF) members TNFSF11/RANKL, TNFSF12/TWEAK, TNFSF13,            TNFSF13B/BAFF/BLys, TNFSF14, TNFSF15, interleukins (IL)            IL-1α, IL-16, IL-2, IL-5, IL-6, IL-10, IL-12, IL-17B,            IL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-22, IL-23, IL-26,            IL-32, 1L33 and IL-34; interferons INFα and INFγ;            granulocyte colony-stimulating factor (GCSF), granulocyte            colony-stimulating factor (GM-CSF); and        -   inflammatory chemokines and their receptors, including            IL-8/CXCL8, ENA-78/CXCL5, GROα/CXCL1, CTAP-III/CXCL7,            IP-10/CXCL10, Mig/CXCL9, PF4/CXCL4, GCP-2/CXCL6, MCP-1/CCL2,            MIP-1α/CCL3, MIP-3α/CCL20, RANTES/CCL5; lymphotactin/XCL1            and fractalkine/CX3CL1.

In one embodiment, the antigen is selected from the group consisting ofTNF, IL-1β, IL-6, IL-17F and IL-23.

In one embodiment, said antibody or fragment thereof is selected fromthe group consisting of visilizumab, otelixizumab, ipilimumab,tremelimumab, pembrolizumab, nivolumab, pidilizumab, MPDL3280A,MEDI-4736, MPDL3280A and lirilumab, and antigen-binding fragmentsthereof.

In one particular embodiment, said antigen is TNF. In one embodiment,said antibody or fragment thereof is selected from the group consistingof adalimumab, infliximab, golimumab, certolimumab pegol, and antigenbinding fragments thereof. In another embodiment said antibody orfragment thereof is a full-length antibody selected from the groupconsisting of adalimumab, infliximab, golimumab and certolimumab pegol.In one particular embodiment, said antibody or antigen binding fragmentthereof is adalimumab or an antigen binding fragment thereof, forexample full-length adalimumab.

The complex as described herein may for example be present in the formof a fusion protein or a conjugate. Thus, said at least one IL-17Abinding polypeptide and said at least one antibody, or antigen bindingfragment thereof, may be coupled by means of chemical conjugation (usingknown organic chemistry methods) or by any other means, such asexpression of the complex as a fusion protein or joined in any otherfashion, either directly or via a linker, for example an amino acidlinker.

Thus, in one embodiment, there is provided a complex as defined herein,wherein said complex is a fusion protein or a conjugate. In oneembodiment, said complex is a fusion protein. In another embodiment,said complex is a conjugate. In one embodiment of said complex, saidIL-17A binding polypeptide is attached to the N-terminus or C-terminusof the heavy chain of said antibody or antigen binding fragment thereof.In another embodiment, said IL-17A binding polypeptide is attached tothe N-terminus or C-terminus of the light chain of said antibody orantigen binding fragment thereof. In one embodiment, said IL-17A bindingpolypeptide is attached to the N-terminus and/or C-terminus of the lightchain and heavy chain of said antibody or antigen binding fragmentthereof. For example, the IL-17A binding polypeptide may be attached toonly the N-terminus of the heavy chain(s), only the N-terminus of thelight chain(s), only the C-terminus of the heavy chain(s), only theC-terminus of the light chain(s), both the N-terminus and the C-terminusof the heavy chain(s), both the N-terminus and the C-terminus of thelight chain(s), only the C-terminus of the light chain(s) and theN-terminus of the heavy chain(s), only the C-terminus of the heavychain(s) and the N-terminus of the light chain(s), of said antibody orantigen binding fragment thereof.

As the skilled person understands, the construction of a fusion proteinoften involves use of linkers between functional moieties to be fused.The skilled person is aware of different kinds of linkers with differentproperties, such as flexible amino acid linkers, rigid amino acidlinkers and cleavable amino acid linkers. Linkers have been used to forexample increase stability or improve folding of fusion proteins, toincrease expression, improve biological activity, affinity and/orbinding, enable targeting and alter pharmacokinetics of fusion proteins.Thus, in one embodiment, the IL-17A binding polypeptide, fusion protein,conjugate or complex as defined herein further comprises at least onelinker. The linker may for example be selected from the group consistingof flexible amino acid linkers, rigid amino acid linkers and cleavableamino acid linkers. Alternatively, the linker may be a non-peptidiclinker. In one embodiment of a fusion protein or conjugate as disclosedherein, said linker is arranged between a first moiety consisting of anIL-17A binding polypeptide as defined herein and a second moietyconsisting of a polypeptide having a desired biological activity. In oneembodiment of a complex as disclosed herein, said linker is arrangedbetween said IL-17A binding polypeptide and said antibody or antigenbinding fragment thereof. The skilled person will appreciate that thepresence of linker arranged in any of above mentioned contexts does notexclude the presence of additional linkers in the same or any othercontext.

Flexible linkers are often used when the joined domains require acertain degree of movement or interaction, and may be particularlyuseful in some embodiments of the IL-17A binding polypeptide, fusionprotein, conjugate or complex as defined herein. Such linkers aregenerally composed of small, non-polar (for example G) or polar (forexample S or T) amino acids. Some flexible linkers primarily consist ofstretches of G and S residues, for example (GGGGS(SEQ ID NO:1301))_(p)and (SSSSG(SEQ ID NO:1302))_(p). Adjusting the copy number “p” allowsfor optimization of the linker in order to achieve appropriateseparation between the functional moieties or to maintain necessaryinter-moiety interaction. Apart from G and S linkers, other flexiblelinkers are known in the art, such as G and S linkers containingadditional amino acid residues, such as T, A, K and E, to maintainflexibility, as well as polar amino acid residues to improve solubility.

In one embodiment, said linker is a flexible linker comprising glycine(G), serine (S) and/or threonine (T) residues. In one embodiment, saidlinker comprises a sequence with a general formula selected from(G_(n)S_(m))_(p) and (S_(n)G_(m))_(p), wherein, independently, n=1-7,m=0-7, n+m 8 and p=1-10. In one embodiment, n=1-5. In one embodiment,m=0-5. In one embodiment, p=1-5. In a more specific embodiment, n=4, m=1and p=1-4. In one embodiment, said linker comprises a sequence selectedfrom the group consisting of G₄S(SEQ ID NO:1301), (G₄S)₂ (SEQ IDNO:1303), (G₄S)₃ (SEQ ID NO:1304) and (G₄S)₄ (SEQ ID NO:1305).

In one embodiment, said linker comprises a sequence with the generalformula GT(G_(n)S_(m))_(p), wherein, independently, n=1-7, m=0-7, n+m 8and p=1-10. In one embodiment, n=1-5. In one embodiment, m=0-5. In oneembodiment, p=1-5. In a more specific embodiment, n=4, m=1 and p=1-4.Thus, in one embodiment wherein n=4, said linker comprises GT(G₄S(SEQ IDNO:1301))_(p). In one specific embodiment, n=4 and p=1, so that saidlinker comprises GTG₄S (SEQ ID NO:1306). In one embodiment, said linkercomprises a sequence selected from the group consisting of GT(G₄S[SEQ IDNO:1301])_(p)TS, GT(G₄S[SEQ ID NO:1301])_(p)PR and GT(G₄S[SEQ IDNO:1301])_(p)PK.

In one embodiment, said linker comprises a sequence with the generalformula GAP(G_(n)S_(m))_(p), wherein, independently, n=1-7, m=0-7, n+m 8and p=1-10. In one embodiment, n=1-5. In one embodiment, m=0-5. In oneembodiment, p=1-5. In a more specific embodiment, n=4, m=1 and p=1-4.Thus, in one embodiment wherein n=4, said linker comprises GAP(G₄S)_(p).In one specific embodiment, n=4 and p=1, so that said linker comprisesGAPG₄S (SEQ ID NO:1307). In one embodiment, said linker comprises asequence selected from the group consisting of GAP(G4S[SEQ IDNO:1301])_(p)TS, GAP(G₄S[SEQ ID NO:1301])_(p)PR and GAP(G₄S[SEQ IDNO:1301])_(p)PK.

In one embodiment, said linker comprises a sequence selected from thegroup consisting of KL(G₄S[SEQ ID NO:1301])_(p), LQ(G₄S[SEQ IDNO:1301])_(p) and YV(G₄S[SEQ ID NO:1301])_(p)PK. In one embodiment, saidlinker comprises a sequence selected from the group consisting ofS₄G(SEQ ID NO:1302), (S₄G)₃ (SEQ ID NO:1308), (S₄G)₄ (SEQ ID NO:1309)and (S₄G)₈ (SEQ ID NO:1310) In one embodiment, said linker comprises asequence selected from the group consisting of VDGS(SEQ ID NO:1311),ASGS(SEQ ID NO:1312) and VEGS(SEQ ID NO:1313). In a specific embodiment,said linker comprises ASGS(SEQ ID NO:1312).

With regard to the description above of fusion proteins, conjugates orcomplexes incorporating an IL-17A binding polypeptide according to thedisclosure, it is to be noted that the designation of first, second andfurther moieties is made for clarity reasons to distinguish betweenIL-17A binding polypeptide or polypeptides according to the invention onthe one hand, and moieties exhibiting other functions on the other hand.These designations are not intended to refer to the actual order of thedifferent domains in the polypeptide chain of the fusion protein,conjugate or complex. Thus, for example, said first moiety may withoutrestriction appear at the N-terminal end, in the middle, or at theC-terminal end of the fusion protein, conjugate or complex.

The disclosure furthermore encompasses polypeptides in which the IL-17Abinding polypeptide according to the first aspect, the IL-17A bindingpolypeptide as comprised in a fusion protein or conjugate according tothe second aspect or in a complex according to the third aspect, furthercomprises a label, such as a label selected from the group consisting offluorescent dyes and metals, chromophoric dyes, chemiluminescentcompounds and bioluminescent proteins, enzymes, radionuclides andparticles. Such labels may for example be used for detection of thepolypeptide.

In embodiments where the labeled IL-17A binding polypeptide comprises anIL-17A binding polypeptide according to the first aspect of thedisclosure and a label, this labeled polypeptide may be used forindirect labeling of IL-17A expressing cells, such as cells ofinflammation-associated cancers. Non-limiting examples ofinflammation-associated cancers include gastric cancers, colorectalcancers, non-small cell lung cancers, hepatocellular carcinomas andadenocarcinomas (Wu et al., 2014 Tumour Biol. 35(6):5347-56; Wu et al.,2012 PLoS One 7(12); Zhang et al. 2012 Asian Pac J Cancer Prev13(8):3955-60; Liu et al., 2011 Biochem Biophys ResCommun.407(2):348-54).

In other embodiments, the labeled IL-17A binding polypeptide is presentas a moiety in a fusion protein, conjugate or complex also comprising asecond and possible further moiety having a desired biological activity.The label may in some instances be coupled only to the IL-17A bindingpolypeptide, and in some instances both to the IL-17A bindingpolypeptide and to the second moiety of the fusion protein or conjugateand/or the antibody or antigen binding fragment of the complex.Furthermore, it is also possible that the label may be coupled to asecond moiety, or antibody or antigen binding fragment thereof only andnot to the IL-17A binding moiety. Hence, in yet another embodiment,there is provided an IL-17A binding polypeptide comprising a secondmoiety, wherein said label is coupled to the second moiety only. Inanother embodiment, there is provided a complex as defined herein,wherein said label is coupled to the antibody or antigen bindingfragment thereof only.

In one embodiment, said IL-17A binding polypeptide, fusion protein,conjugate or complex as described herein comprises a chelatingenvironment provided by a polyaminopolycarboxylate chelator conjugatedto the IL-17A binding polypeptide via a thiol group of a cysteineresidue or an amine group of a lysine residue.

In embodiments where the IL-17A binding polypeptide, fusion protein,conjugate or complex is radiolabeled, such a radiolabeled polypeptidemay comprise a radionuclide. A majority of radionuclides have a metallicnature and metals are typically incapable of forming stable covalentbonds with elements presented in proteins and peptides. For this reason,labeling of proteins and peptides with radioactive metals is performedwith the use of chelators, i.e. multidentate ligands, which formnon-covalent compounds, called chelates, with the metal ions. In anembodiment of the IL-17A binding polypeptide, fusion protein, conjugateor complex, the incorporation of a radionuclide is enabled through theprovision of a chelating environment, through which the radionuclide maybe coordinated, chelated or complexed to the polypeptide.

One example of a chelator is the polyaminopolycarboxylate type ofchelator. Two classes of such polyaminopolycarboxylate chelators can bedistinguished: macrocyclic and acyclic chelators.

In one embodiment, the IL-17A binding polypeptide, fusion protein orconjugate comprises a chelating environment provided by apolyaminopolycarboxylate chelator conjugated to the IL-17A bindingpolypeptide via a thiol group of a cysteine residue or an epsilon aminegroup of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes ofindium, gallium, yttrium, bismuth, radioactinides and radiolanthanidesare different derivatives of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In oneembodiment, a chelating environment of the IL-17A polypeptide, fusionprotein, conjugate or complex is provided by DOTA or a derivativethereof. More specifically, in one embodiment, the chelatingpolypeptides encompassed by the present disclosure are obtained byreacting the DOTA derivative1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with saidpolypeptide.

Additionally, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) andderivatives thereof may be used as chelators. Hence, in one embodiment,there is provided an IL-17A binding polypeptide, fusion protein,conjugate or complex, wherein the polyaminopolycarboxylate chelator is1,4,7-triazacyclononane-1,4,7-triacetic acid or a derivative thereof.

The most commonly used acyclic polyaminopolycarboxylate chelators aredifferent derivatives of DTPA (diethylenetriamine-pentaacetic acid).Hence, polypeptides having a chelating environment provided bydiethylenetriaminepentaacetic acid or derivatives thereof are alsoencompassed by the present disclosure.

In one embodiment, said IL-17A binding polypeptide, fusion protein,conjugate or complex as described herein further comprises one or morepolyethylene glycol (PEG) moieties, e.g. in order to improvepharmacokinetic properties of the molecule.

In a fourth aspect of the present disclosure, there is provided apolynucleotide encoding an IL-17A binding polypeptide or a fusionprotein as described herein; an expression vector comprising saidpolynucleotide; and a host cell comprising said expression vector.

Also encompassed by this disclosure is a method of producing apolypeptide or fusion protein as described above, comprising culturingsaid host cell under conditions permissive of expression of saidpolypeptide from its expression vector, and isolating the polypeptide.

The IL-17A binding polypeptide of the present disclosure mayalternatively be produced by non-biological peptide synthesis usingamino acids and/or amino acid derivatives having protected reactiveside-chains, the non-biological peptide synthesis comprising

-   -   step-wise coupling of the amino acids and/or the amino acid        derivatives to form a polypeptide according to the first aspect        having protected reactive side-chains,    -   removal of the protecting groups from the reactive side-chains        of the polypeptide, and    -   folding of the polypeptide in aqueous solution.

It should be understood that the IL-17A binding polypeptide according tothe present disclosure may be useful as a therapeutic, diagnostic orprognostic agent in its own right or as a means for targeting othertherapeutic or diagnostic agents, with e.g. direct or indirect effectson IL-17A. A direct therapeutic effect may for example be accomplishedby inhibiting IL-17A signaling, such as by blocking IL-17A from bindingto one or more of its receptors.

In another aspect, there is provided a composition comprising an IL-17Abinding polypeptide, fusion protein, conjugate or complex as describedherein and at least one pharmaceutically acceptable excipient orcarrier. In one embodiment, said composition further comprises at leastone additional active agent, such as at least two additional activeagents, such as at least three additional active agents. Non-limitingexamples of additional active agents that may prove useful in such acomposition are the therapeutically active polypeptides, immune responsemodifying agents and toxic compounds described herein.

The skilled person will appreciate that said IL-17A binding polypeptide,fusion protein, conjugate or complex, or a pharmaceutical compositioncomprising an anti-IL-17A binding polypeptide, fusion protein, conjugateor complex as described herein may be administered to a subject usingstandard administration techniques, such as including oral, topical,intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal,intramuscular, intranasal, buccal, sublingual or suppositoryadministration. Thus, in one embodiment there is provided an IL-17Abinding polypeptide, fusion protein, conjugate or complex or apharmaceutical composition as described herein for oral, topical,intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal,intramuscular, intranasal, buccal, sublingual or suppositoryadministration. In one particular embodiment there is provided an IL-17Abinding polypeptide, fusion protein, conjugate or complex or apharmaceutical composition as described herein for oral administration.In another particular embodiment there is provided an IL-17A bindingpolypeptide, fusion protein, conjugate or complex or a pharmaceuticalcomposition as described herein for topical administration, such astopical administration to the eye.

IL-17A may also serve as a valuable marker to for diagnosis andprognosis of certain cancers, such as inflammation-associated cancersfor example gastric cancers, colorectal cancers, non-small cell lungcancers, hepatocellular carcinomas and adenocarcinomas. For example,IL-17 has been linked to the prognosis and poor survival in patientssuffering from colorectal carcinoma and hepatocellular carcinoma.

Hence, in another aspect of the present disclosure, there is provided anIL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition as described herein for use as a medicament, a diagnosticagent or a prognostic agent.

In one embodiment, there is provided an IL-17A binding polypeptide,fusion protein, conjugate, complex or composition as described herein,for use as a medicament to modulate IL-17A function in vivo. As usedherein, the term “modulate” refers to changing the activity, such asrendering IL-17A function hypomorph, partially inhibiting or fullyinhibiting IL-17A function.

Non-limiting examples of IL-17A associated conditions or diseases,wherein the IL-17A binding polypeptides may be useful for treatment,prognosis and/or diagnosis of include arthritis, such as rheumatoidarthritis, arthritis chronica progrediente, arthritis deformans andrheumatic diseases, diseases involving bone loss, inflammatory pain,spondyloarhropathies including ankylosing spondylitis, Reiter syndrome,reactive arthritis, psoriatic arthritis, enterophathc arthritis,pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis,systemic onset rheumatoid arthritis, osteoarthritis; hypersensitivityconditions, such as hypersensitivity (including both airwayshypersensitivity and dermal hypersensitivity), allergies and responsesto allergen exposure; gastrointestional conditions or diseases, such asautoimmune inflammatory bowel disease (including e.g. ulcerativecolitis, Crohn's disease and Irritable Bowel Syndrome), celiac disease(idiopathic sprue), intraperitoneal abscesses and adhesions, primarybiliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, viralhepatitis, chronic active hepatitis and Helicobacter pylori associatedgastritis; ophthalmic conditions and diseases, such as endocrineophthalmopathy, Graves disease, uveitis (anterior and posterior),keratoconjunctivitis sicca (dry eye disease), vernalkeratoconjunctivitis, herpetic stromal keratitis and dry eye disease;nephrological conditions and disease, such as glomerulonephritis (withand without nephrotic syndrome, e.g. including idiopathic nephroticsyndrome or minimal change nephropathy); acute conditions and disease,such as acute and hyperacute inflammatory reactions, septic shock (e.g.,endotoxic shock and adult respiratory distress syndrome), meningitis,pneumonia, severe burns, acute infections, septicemia, stroke andischemic; cachexia (wasting syndrome), such as cachexia associated withmorbid TNF release, cachexia consequent to infection, cachexiaassociated with cancer, cachexia associated with organ dysfunction andAIDS-related cachexia; bone related conditions and diseases, such asdiseases of bone metabolism including osteoarthritis, osteoporosis,inflammatory arthritides, bone loss such as age-related bone loss,periodontal disease, loosening of bone implants and bone erosion;juvenile conditions and diseases, such as juvenile rheumatoid arthritis,pauciarticular juvenile rheumatoid arthritis, polyarticular juvenilerheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,juvenile ankylosing spondylitis, juvenile enteropathic arthritis,juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome(Seronegativity, Enthesopathy, Arthropathy Syndrome), juveniledermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma,juvenile systemic lupus erythematosus and juvenile vasculitis;vasculitis including vasculitis of large vessels, such as Polymyalgiarheumatica, Takayasu's arteritis and Temporal arteritis, vasculitis ofmedium vessels, such as Buerger's disease, Cutaneous vasculitis,Kawasaki disease and Polyarteritis nodosa, vasculitis of small vessels,such as Behçet's syndrome, Churg-Strauss syndrome, cutaneous vasculitis,Henoch-Schönlein purpura, microscopic polyangiitis, Wegener'sgranulomatosis and Golfer's vasculitis, vasculitis of variable vesselsand arteritis; dermatological conditions or diseases, such as psoriasis,plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis, erythrodermic psoriasis, dermatitis and atopic dermatitis;pulmonary conditions and diseases, such as obstructive or inflammatoryairways diseases, asthma, bronchitis, COPD, pneumoconiosis, pulmonaryemphysema, acute and hyperacute inflammatory reactions, interstitiallung fibrosis, airway inflammation and bronchial asthma; metabolicconditions and diseases, such as atherosclerosis, dyslipidemia and TypeI diabetes mellitus; as well as other systemic autoimmune conditions anddiseases, such as systemic lupus erythematosus (SLE), lupus nephritis,polychondritis, myasthenia gravis, Steven-Johnson syndrome, tumors,myositis, dermatomyositis, adult-onset Still's disease, polymyalgiarheumatica, sarcoidosis, scleroderma, sclerosis, systemic sclerosis,Sjogren's syndrome, multiple sclerosis (MS), Guillain-Barre disease,Addison's disease and Raynaud's phenomenon.

The IL-17A binding polypeptides as disclosed herein may furthermore beuseful for the treatment of recipients of heart, lung, combinedheart-lung, liver, kidney, pancreatic, skin or corneal transplants,including allograft rejection or xenograft rejection, and for theprevention of graft-versus-host disease, such as following bone marrowtransplant, and organ transplant associated arteriosclerosis.

The IL-17A binding polypeptides as disclosed herein may furthermore beuseful for the treatment, diagnosis or prognosis ofinflammation-associated cancers. The term “cancer” as used herein refersto tumor diseases and/or cancer, such as metastatic or invasive cancers,for example lung cancer, non small cell lung (NSCL) cancer,bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, cutaneous or intraocularmelanoma, rectal cancer, cancer of the anal region, stomach cancer,gastric cancer, colon cancer, colorectal cancer, cancer of the smallintestines, esophageal cancer, liver cancer, pancreas cancer, breastcancer, ovarian cancer, uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, bladder cancer,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular carcinoma, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma, adenocarcinomas, lymphoma, lymphocytic leukemia, orcancer of unknown origin, or other hyperplastic or neoplastic IL-17Aassociated condition, including refractory versions of any of the abovecancers or a combination of one or more of the above cancers orhyperproliferative diseases.

Non-limiting examples of inflammation-associated cancers include gastriccancers, colorectal cancers, non-small cell lung cancers, hepatocellularcarcinomas and adenocarcinomas.

In one embodiment, there is provided an IL-17A binding polypeptide,fusion protein, conjugate, complex or composition for use in thetreatment, diagnosis or prognosis of an IL-17A associated condition,such as a condition selected from the group consisting of inflammatorydiseases, autoimmune diseases and cancer, such as inflammatory diseasesand autoimmune diseases. In one embodiment, said condition is selectedfrom the group consisting of inflammatory conditions, allergicconditions, hypersensitivity reactions, autoimmune diseases, severeinfections and transplant rejections.

In one particular embodiment, said IL-17A associated condition isselected from the group consisting of rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, psoriasis, multiple sclerosis,systemic lupus erythematosus, uveitis and dry eye disease.

In an even more particular embodiment, said IL-17A associated conditionis psoriasis.

In another embodiment, said IL-17A associated condition is aninflammation-associated cancer, such as a cancer selected from the groupconsisting of gastric cancers, colorectal cancers, non-small cell lungcancers, hepatocellular carcinomas and adenocarcinomas.

In a related aspect, there is provided a method of detecting IL-17A,comprising providing a sample suspected to contain IL-17A, contactingsaid sample with an IL-17A binding polypeptide, fusion protein,conjugate, complex or composition as described herein, and detecting thebinding of the IL-17A binding polypeptide, fusion protein, conjugate,complex or composition to indicate the presence of IL-17A in the sample.In one embodiment, said method further comprises an intermediate washingstep for removing non-bound polypeptide, fusion protein, conjugate,complex or composition, after contacting the sample.

In one embodiment, said method is a diagnostic or prognostic method, fordetermining the presence of IL-17A in a subject, the method comprisingthe steps:

-   -   contacting the subject, or a sample isolated from the subject,        with an IL-17A binding polypeptide, fusion protein, conjugate,        complex or composition as described herein, and    -   obtaining a value corresponding to the amount of the IL-17A        binding polypeptide, fusion protein, conjugate, complex or        composition that has bound in said subject or to said sample.

In one embodiment, said method further comprises an intermediate washingstep for removing non-bound polypeptide, fusion protein, conjugate,complex or composition, after contacting the subject or sample andbefore obtaining a value.

In one embodiment, said method further comprises a step of comparingsaid value to a reference. Said reference may be scored by a numericalvalue, a threshold or a visual indicator, for example based on a colorreaction. The skilled person will appreciate that different ways ofcomparison to a reference are known in the art may be suitable for use.

In one embodiment of such a method, said subject is a mammalian subject,such as a human subject.

In one embodiment, said method is performed in vivo.

In one embodiment, said method is performed in vitro.

In a related aspect, there is provided a method of treatment of anIL-17A associated condition, comprising administering to a subject inneed thereof an effective amount of an IL-17A binding polypeptide,fusion protein, conjugate, complex or composition as described herein.In a more specific embodiment of said method, the IL-17A bindingpolypeptide, fusion protein, conjugate, complex or composition asdescribed herein modulates IL-17A function in vivo.

In one embodiment, said IL-17A associated condition is selected from thegroup consisting of inflammatory diseases, autoimmune diseases andcancer, such as a group consisting of inflammatory diseases andautoimmune diseases. In one particular embodiment of said aspect, theIL-17A associated condition is selected from the group consisting ofinflammatory conditions, allergic conditions, hypersensitivityreactions, autoimmune diseases, severe infections and transplantrejections. In one embodiment, said IL-17A associated condition isselected from the group consisting of rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, psoriasis, multiple sclerosis,systemic lupus erythematosus, uveitis and dry eye disease. In a morespecific embodiment, said IL-17A associated condition is psoriasis. Inanother embodiment, said IL-17A associated condition is cancer, such asa cancer selected from the group consisting of gastric cancers,colorectal cancers, non-small cell lung cancers, hepatocellularcarcinomas and adenocarcinomas.

It may be beneficial to administer a therapeutically effective amount ofan IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition as described herein together with least one second drugsubstance, such as an immune response modifying agent, toxic compound oran anti-cancer agent.

As used herein, the term “co-adminstration” encompasses concomitantadminstration and adminstration in sequence. Thus, in one embodiment,there is provided a method as defined above, further comprisingco-administration of an immune response modulating agent. In anotherembodiment, there is provided a method as defined above, furthercomprising co-administration of an additional anti-inflammatory agent.In another embodiment, there is provided a method as defined above,further comprising co-administration of a toxic compound. In anotherembodiment, there is provided a method as defined above, furthercomprising co-administration of an anti-cancer agent.

Non-limiting examples of immune response modulating agents and toxiccompounds are given above. Non-limiting examples of anti-cancer agentsinclude agents selected from the group consisting of auristatin,anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin,the CC-1065 anti-tumorantibiotic, ecteinsascidin, geldanamycin,maytansinoid, methotrexate, mycotoxin, taxol, ricin, bouganin, gelonin,pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and theiranalogues, and derivates thereof and combinations thereof. A skilledperson would appreciate that the non-limiting examples of cytotoxicagents include all possible variant of said agents, for example theagent auristatin includes for example auristatin E, auristatin F,auristatin PE, and derivates thereof.

While the invention has been described with reference to variousexemplary aspects and embodiments, it will be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted for elements thereof without departing from the scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation or molecule to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to any particular embodimentcontemplated, but that the invention will include all embodimentsfalling within the scope of the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1QQ are a listing of the amino acid sequences of examples ofIL-17A binding polypeptides of the present disclosure (SEQ IDNO:1-1222), a control polypeptide (SEQ ID NO:1223), the albumin bindingpolypeptides PP013 (SEQ ID NO:1224) and PEP07843 (SEQ ID NO:1225) aswell as the amino acid sequences of human IL-17A (SEQ ID NO:1226),murine IL-17A (SEQ ID NO:1227), cynomolgus monkey IL-17A (SEQ IDNO:1229), rhesus monkey IL-17A (SEQ ID NO:1230) and human IL-17F (SEQ IDNO:1228) used for selection, screening and/or characterization forillustration of the invention. In the IL-17A binding polypeptides of thepresent disclosure, the deduced IL-17A binding motifs (BMs) extend fromresidue 8 to residue 36 in each sequence. The amino acid sequences ofthe 49 amino acid residues long polypeptides (BMod) predicted toconstitute the complete three-helix bundle within each of these Zvariants extend from residue 7 to residue 55.

FIG. 2 shows inhibition of IL-17A induced IL-6 production assessed bythe NHDF assay described in Example 3. (A) His₆-Z06260 (open dimonds),His₆-Z06282 (open squares) and His₆-Z06455 (open circles) were titratedin a medium containing IL-17A. (B) Z06260-ABD (closed diamonds),His₆-Z06282 (open squares), Z06282-ABD (filled squares), His₆-Z06455(open circles) and Z06455-ABD (filled circles) were titrated in a mediumcontaining IL-17A and HSA.

FIG. 3 shows the IL-17A binding capacity of a set of Z variants from thefirst and second maturation library assayed by ELISA as described inExample 8. The results are displayed as percent binding capacitycompared to the IL-17A binding variant Z10241 (SEQ ID NO:11).

FIGS. 4A-4C shows the result of binding specificity analysis performedin a Biacore instrument as described in Example 8. Sensorgrams wereobtained by injection of 20 nM of the His₆-tagged Z variants Z10508 (SEQID NO:2) (A), Z10532 (SEQ ID NO:1) (B) and Z15167 (SEQ ID NO:4) (C) overhuman IL-17A (black), human IL-17A/F (dark grey) and human IL-17F (lightgrey), respectively, immobilized on the surface of a CM5 chip.

FIG. 5 shows inhibition of IL-17A induced IL-6 production for aselection of Z variants originating from the first maturation selection(solid curves) compared to one binder from the primary selection (brokencurve) assayed as described in Example 8. All binders inhibited IL-17Ain a dose dependent manner and the maturated binders had an increasedblocking capacity compared to the primary binder.

FIGS. 6A-6C show binding of human IL-17A (grey) and cynomolgus IL-17A(black) to (A) ZAZ3363 (SEQ ID NO:1244), (B) ZAZ3364 (SEQ ID NO:1245)and (C) ZAZ3365 (SEQ ID NO:1246), analyzed in a Biacore instrument asdescribed in Example 11. The resulting curves, from which responses froma blank surface were subtracted, correspond to injections of therespective IL-17A protein at concentrations 2.5, 10 and 40 nM.

FIGS. 7A-7B show inhibition of IL-17A induced IL-6 production in theNHDF assay described in Example 14. FIG. 7A shows a superior inhibitoryefficacy with the dimeric Z-ABD-Z polypeptide ZAZ3220 (SEQ ID NO:1236,solid black line) compared to the corresponding monomeric Z variantZ10241 (SEQ ID NO:11; dotted black line). FIG. 7B shows an evaluation ofdifferent linker lengths between the Z and the ABD moieties in Z-ABD-Zpolypeptides comprising the Z variant Z06282 (SEQ ID NO:1206). TheZ-ABD-Z polypeptides ZAZ3174 (SEQ ID NO:1233), ZAZ3176 (SEQ ID NO:1235),ZAZ3234 (SEQ ID NO:1238), ZAZ3235 (SEQ ID NO:1239), ZAZ3236 (SEQ IDNO:1240) and ZAZ3237 (SEQ ID NO:1241) with various numbers of G₄Srepeats, or minimal linkers, on each side of the ABD were compared.

FIGS. 8A-8B show dose dependent inhibition by Z-ABD-Z polypeptides inthe hIL-17A induced KC-model described in Example 15. (A) Completeinhibition of KC production by ZAZ3174 (SEQ ID NO:1233) was obtained ata dose of 2.5 mg/kg. (B) Complete inhibition of KC production by ZAZ3220(SEQ ID NO:1236) was obtained ata dose of 0.4 mg/kg. Doses indicated onthe x-axis are given in mg/kg and the corresponding pmol dose. Thenumbers above the bar indicate percent inhibition in each dose group;72% inhibition was obtained with 0.1 mg/kg ZAZ3220. LOQ=limit ofquantification.

FIGS. 9A-9B show the pharmacokinetic profiles of the Z-ABD-Zpolypeptides (A) ZAZ3220 (SEQ ID NO:1236) and (B) ZAZ3363 (SEQ IDNO:1244), following a single i.v. (black line) or s.c. (gray brokenline) administration to SD rats as described in Example 16. The meanserum concentrations versus time is displayed.

FIG. 10 shows the result of topical administration to the eyes ofrabbits performed as described in Example 17. Uptake of Z variant Z10199(SEQ ID NO:1217) and the Z-ABD-Z polypeptide ZAZ3174 (SEQ ID NO:1233)was demonstrated both in aqueous humor and vitreous humor, whereas thecontrol IgG antibody did not penetrate the eye.

FIGS. 11A-11B show the activity of ZAZ3363 (SEQ ID NO:1244) formulatedin OAF1 and OAF2, respectively, in comparison to formulation in PBS, andanalyzed in the NHDF assay described in Example 18. (A) OAF1 (filledcircles), PBS (crosses) and OAF1 without ZAZ3363 (open diamonds). (B)OAF2 (open triangles), PBS (crosses) and OAF2 without ZAZ3363 (opendiamonds).

FIG. 12 shows the pharmacokinetic profile of intraduodenaladministration of ZAZ3363 formulated in OAF1 (filled circles), OAF2(open triangles) and PBS (crosses) performed as described in Example 18.

FIGS. 13A-13D show the evaluation of the complexes HC_(Ada)-Z14253 andLC_(Ada)-Z14253 in the NHDF assay described in Example 19. (A) Schematicof the complexes HC_(Ada)-Z14253 (left) and LC_(Ada)-Z14253 (right). (B)Inhibition of IL-17A induced IL-6 production. (C) Inhibition ofTNF-induced IL-8 production. (D) Inhibition of TNF and IL-17A-inducedIL-8 production. Z04726-ABD is a negative control, included in all threeassays.

FIGS. 14A-14B show the pharmacokinetic profiles of the Z-ABD-Zpolypeptide ZAZ3363 (SEQ ID NO:1244) in cynomolgus monkeys followingi.v. administration of 20 mg/kg (black) or 40 mg/kg (grey) on day 1, 4,7 and 10 as described in Example 20. Mean plasma concentrations versustime are shown following analysis of (A) the first injection on day 1and (B) the fourth injection on day 10. Error bars represent standarddeviation.

FIG. 15 shows the pharmacokinetic profiles of the Z-ABD-Z polypeptideZAZ3363 (SEQ ID NO:1244) in individual beagle dogs following oraladministration. 150 mg of ZAZ3363 was administered as enteric coatedcapsules on day 0.

EXAMPLES Summary

The following Examples disclose the development of novel Z variantmolecules targeted to interleukin 17A (IL-17A) based on phage displaytechnology. The IL-17A binding polypeptides described herein weresequenced, and their amino acid sequences are listed in FIGS. 1A-1QQwith the sequence identifiers SEQ ID NO:1-1222. The Examples furtherdescribe the characterization of IL-17A binding polypeptides and invitro and in vivo functionality of said polypeptides.

Example 1 Selection and Screening of IL-17A Binding Z Variants Materialsand Methods

Biotinylation of target protein: Human IL-17A (hIL-17A Peprotech cat.no. 200-17; SEQ ID NO:1226) and murine IL-17A (mIL-17A Peprotech cat.no. 210-17; SEQ ID NO:1227) were biotinylated according to themanufacturer's recommendations at room temperature (RT) for 30 min usingNo-Weigh EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scentific, cat. no. 21327)at a 20× molar excess. Subsequent buffer exchange to phosphate bufferedsaline (PBS,10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) wasperformed using a dialysis cassette (Slide-a-lyzer 3.5 K, 3500 MWCO,Thermo Scentific, cat. no. 66333) according to the manufacturer'sinstructions.

Phage display selection of IL-17A binding Z variants: A library ofrandom variants of protein Z displayed on bacteriophage, constructed inphagemid pAY02592 essentially as described in Grönwall et al. (2007) JBiotechnol, 128:162-183, was used to select IL-17A binding Z variants.In this library, an albumin binding domain (abbreviated ABD andcorresponding to GA3 of protein G from Streptococcus strain G148) isused as fusion partner to the Z variants. The library is denotedZlib006Naive.II and has a size of 1.5×10¹⁰ library members (Z variants).E. coli RRIΔM15 cells (Ruther et al., (1982) Nucleic Acids Res10:5765-5772) from a glycerol stock containing the phagemid libraryZlib006Naive.II, were inoculated in 20 l of a defined proline freemedium [3 g/l KH₂PO₄, 2 g/l K₂HPO₄, 0.02 g/l uracil, 6.7 g/l YNB (Difco™Yeast Nitrogen Base w/o amino acids, Becton Dickinson), 5.5 g/l glucosemonohydrate, 0.3 g/l L-alanine, 0.24 g/l L-arginine monohydrochloride,0.11 g/l L-asparagine monohydrate, 0.1 g/l L-cysteine, 0.3 g/lL-glutamic acid, 0.1 g/l L-glutamine, 0.2 g/l glycine, 0.05 g/lL-histidine, 0.1 g/l L-isoleucine, 0.1 g/l L-leucine, 0.25 g/l L-lysinemonohydrochloride, 0.1 g/l L-methionine, 0.2 g/l L-phenylalanine, 0.3g/l L-serine, 0.2 g/l L-threonine, 0.1 g/l L-tryptophane, 0.05 g/lL-tyrosine, 0.1 g/l L-valine], supplemented with 100 μg/ml ampicillin.The cultivations were grown at 37° C. in a fermenter (Belach Bioteknik,BR20). When the cells reached an optical density at 600 nm (OD600) of0.75, approximately 2.6 l of the cultivation was infected using a 10×molar excess of M13K07 helper phage (New England Biolabs, cat. no.N0315S). The cells were incubated for 30 min, whereupon the fermenterwas filled up to 20 I with cultivation medium [2.5 g/l (NH₄)₂SO₄; 5.0g/l Yeast Extract (Merck 1.03753.0500); 25 g/l Peptone (Scharlau07-119); 2 g/l K₂HPO₄; 3 g/l KH₂PO₄; 1.25 g/l Na₃C₆H₅O₇.2 H₂O; 0.1 ml/lBreox FMT30 antifoaming agent] supplemented with 100 pMisopropyl-6-D-1-thiogalactopyranoside (IPTG) for induction of expressionand with 50 μg/ml ampicillin, 12.5 μg/ml carbenicillin, 25 μg/mlkanamycin, 35 ml/I of 1.217 M MgSO₄ and 10 ml of a trace elementsolution [129 mM FeCl₃; 36.7 mM ZnSO₄; 10.6 mM CuSO₄; 78.1 mM MnSO4;94.1 mM CaCl₂, dissolved in 1.2 M NCI]. A glucose-limited fed-batchcultivation was started where a 600 g/l glucose solution was fed to thereactor (15 g/h in the start, 40 g/h at the end of the fermentationafter 17 h). Through the automatic addition of 25% NH₄OH, the pH wascontrolled at 7. Air was supplemented (10 l/min) and the stirrer was setto keep the dissolved oxygen level above 30%. The cells in thecultivation were removed by tangential flow filtration.

The phage particles were precipitated from the supernatant twice inPEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolvedin PBS and glycerol as described in Grönwall et al., supra. Phage stockswere stored at −80° C. before use.

Selections against biotinylated hIL-17A (b-hIL-17A) or biotinylatedmIL-17A (b-mIL-17A) were performed in four cycles divided in sixdifferent tracks (Table 2). Phage stock preparation, selection procedureand amplification of phage between selection cycles were performedessentially as described in WO2009/077175 for selection against anothertarget with the following exceptions. Exception 1: PBS supplemented with3% bovine serum albumin (BSA, Sigma, cat. no. A3059) and 0.1% Tween20(Acros Organics, cat. no. 233362500) was used as selection buffer.Exception 2: pre-selection was performed in cycles 1-3 by incubation ofphage stock with Dynabeads® M-280 Streptavidin (SA beads, Invitrogen,cat. no. 11206D). Exception 3: all tubes and beads used in theselections were pre-blocked with PBS supplemented with 5% BSA and 0.1%Tween20. Exception 4: selections were performed in solution at RT andthe time for selection was 200 min in the first cycle and 120 min in thefollowing cycles. Exception 5: this was followed by catching oftarget-phage complexes on SA beads using 1 mg beads per 6.4 μg b-hIL-17Aor b-mIL-17A. Exception 6: E. coli strain XL1-Blue cells (AgilentTechnologies, cat. no. 200268) grown in medium supplemented with 10μg/ml tetracycline (exception 7) were used for infection. Exception 8:tryptone yeast extract plates (15 g/l agar, 10 g/l tryptone water(Merck), 5 g/l yeast extract, 3 g/l NaCl, 2% glucose) supplemented with0.1 g/l ampicillin and 0.01 g/l tetracycline were used for spreading ofbacteria. Exception 9: a 10× excess of M13K07 helper phage compared tobacteria was allowed to infect log phase bacteria.

An overview of the selection strategy, describing an increasedstringency in subsequent cycles obtained using a lowered targetconcentration and an increased number of washes, is shown in Table 2.

TABLE 2 Overview of the selection from a primary library Phage stockTarget Higher amount Selection from library or concentration Number ofof phage into Cycle track selection track Target (nM) washes selectiontrack 1 1 Zlib006Naive.II hIL-17A 50 2 — 2 1-1 1 hIL-17A 25 4 Yes 2 1-21 hIL-17A 25 4 — 2 1-3 1 hIL-17A 5 5 — 2 1-4 1 mIL-17A 50 4 — 3 1-1-11-1 hIL-17A 12.5 6 Yes 3 1-2-1 1-2 hIL-17A 12.5 6 — 3 1-3-1 1-3 hIL-17A1 8 — 3 1-4-1 1-4 hIL-17A 12.5 6 — 3 1-4-2 1-4 mIL-17A 5 8 — 4 1-1-1-11-1-1 hIL-17A 5 8 Yes 4 1-2-1-1 1-2-1 hIL-17A 5 8 — 4 1-3-1-1 1-3-1hIL-17A 0.25 12 — 4 1-3-1-2 1-3-1 hIL-17A 0.05 15 — 4 1-4-1-1 1-4-1mIL-17A 5 8 — 4 1-4-2-1 1-4-2 hIL-17A 0.5 12 —

Track 1 was divided in the second to the fourth cycles, resulting in atotal of four tracks (1-1 to 1-4) in cycle 2, five tracks (1-1-1 to1-4-2) in cycle 3 and six tracks (1-1-1-1 to 1-4-2-1) in cycle 4. Thenumber of phage particles used for selections was about 2000 times thenumber of eluted phage particles in the previous cycle, but a higheramount was used in selection tracks 1-1, 1-1-1 and 1-1-1-1.

Washes were performed for 1 min using PBST 0.1% (PBS supplemented with0.1% Tween-20), and elution was carried out as described inWO2009/077175.

Production of Z variants for ELISA: The Z variants were produced byinoculating single colonies from the selections into 1 ml TSB-YE mediumsupplemented with 100 μg/ml ampicillin and 0.1 mM IPTG in deep-wellplates (Nunc, cat. no. 278752). The plates were incubated for 18-24 h at37° C. Cells were pelleted by centrifugation, re-suspended in 400 μlPBST 0.05% and frozen at −80° C. to release the periplasmic fraction ofthe cells. Frozen samples were thawed in a water bath and thefreeze-thawing procedure was repeated six times. 400 μl PBST 0.05% wasadded to the thawed samples and cells were pelleted by centrifugation.

The final supernatant of the periplasmic extract contained theZ-variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (SEQ ID NO:1314) (Grönwall et al., supra). Z##### refers to individual 58 amino acid residue Z variants.

ELISA screening of Z variants: The binding of Z variants to human IL-17Awas analyzed in ELISA assays. Half-area 96-well ELISA plates (Costar,cat. no. 3690) were coated at 4° C. overnight with 2 μg/ml of ananti-ABD goat antibody (produced in-house) diluted in coating buffer (50mM sodium carbonate, pH 9.6; Sigma, cat. no. C3041). The antibodysolution was poured off and the wells were washed in water and blockedwith 100 μl of PBSC (PBS supplemented with 0.5% casein) for 1 to 3 h atRT. The blocking solution was discarded and 50 μl periplasmic solutionswere added to the wells and incubated for 1.5 h at RT under slowagitation. As a blank control, PBST 0.05% was added instead of theperiplasmic sample. The supernatants were poured off and the wells werewashed 4 times with PBST 0.05%. Next, 50 μl of b-hIL-17A at aconcentration of 6.5 nM in PBSC was added to each well. The plates wereincubated for 1.5 h at RT followed by washes as described above.Streptavidin conjugated HRP (Thermo Scientific, cat. no. N100) diluted1:30,000 in PBSC, was added to the wells and the plates were incubatedfor 1 h. After washing as described above, 50 μl ImmunoPure™ B substrate(Thermo Scientific, cat. no. 34021) was added to the wells and theplates were treated according to the manufacturer's recommendations. Theabsorbance at 450 nm was measured using a Victor³ multi-well platereader (Perkin Elmer).

Sequencing: In parallel with the ELISA screening, all clones were pickedfor sequencing. PCR fragments were amplified from single colonies,sequenced and analyzed essentially as described in WO2009/077175.

Results

Phage display selection of IL-17A binding Z variants: Individual cloneswere obtained after four cycles of phage display selections againstb-hIL-17A and b-mIL-17A.

ELISA screening of Z variants: The clones obtained after four cycles ofselection were expanded in 96-well plates and screened for hIL-17Abinding activity in ELISA. 42 Z variants were found to give a responseof 4× the blank control or higher (0.43-3.2 AU) against hIL-17A at aconcentration of 6.5 nM. No response was obtained for the blank control.

Sequencing: Sequencing was performed for clones obtained after fourcycles of selection. Each variant was given a unique identificationnumber #####, and individual variants are referred to as Z #####. Theamino acid sequences of the 58 amino acid residues long Z variants arelisted in FIG. 1 and in the sequence listing as SEQ ID NO: 1200-1216.The deduced IL-17A binding motifs extend from residue 8 to residue 36 ineach sequence. The amino acid sequences of the 49 amino acid residueslong polypeptides predicted to constitute the complete three-helixbundle within each of these Z variants extend from residue 7 to residue55.

Example 2 Production of Monomeric IL-17A Binding Z Variants

This Example describes the general procedure for subcloning andproduction of His-tagged Z variants and Z variants in fusion with ABD,which are used throughout in characterization experiments below.

Materials and Methods

Subcloning of Z variants with a His-tag: The DNA of respective Z variantwas amplified from the library vector pAY02592. A subcloning strategyfor construction of monomeric Z variant molecules with N-terminal His₆tag was applied using standard molecular biology techniques (essentiallyas described in WO2009/077175 for Z variants binding another target).The Z gene fragments were subcloned into the expression vector pAY01448resulting in the encoded sequence MGSSHHHHHHLQ-[Z #####]-VD (SEQ IDNO:1315).

Subcloning of Z variants in fusion with ABD: The DNA of respective Zvariant was amplified from the library vector pAY02592. A PCR wasperformed using suitable primer pairs and the resulting gene fragmentswere cleaved with restriction enzymes PstI and AccI and ligated into theexpression vector pAY03362 digested with the same enzymes, resulting inan ABD fusion protein, wherein the ABD variant is PP013 (SEQ IDNO:1224). The constructs encoded by the expression vectors wereMGSSLQ-[Z #####]-VDSS-PP013 (SEQ ID NO:1316).

Cultivation: E. coli BL21(DE3) cells (Novagen) were transformed withplasmids containing the gene fragment of each respective IL-17A bindingZ variant and cultivated at 37° C. in 800 or 1000 ml of TSB-YE mediumsupplemented with 50 μg/ml kanamycin. In order to induce proteinexpression, IPTG was added to a final concentration of 0.2 mM at OD₆₀₀=2and the cultivation was incubated at 37° C. for another 5 h. The cellswere harvested by centrifugation.

Purification of IL-17A binding Z variants with a His₆-tag: Approximately2-5 g of each cell pellet was re-suspended in 30 ml of binding buffer(20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4)supplemented with Benzonase® (Merck) to a concentration of 15 U/ml.After cell disruption, cell debris was removed by centrifugation andeach supernatant was applied on a 1 ml His GraviTrap IMAC column (GEHealthcare). Contaminants were removed by washing with wash buffer (20mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4 and the IL-17Abinding Z variants were subsequently eluted with elution buffer (20 mMsodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4).

Purification of IL-17A binding Z variants in fusion with ABD:Approximately 1-2 g of each cell pellet was re-suspended in 30 mlTST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH8.0) supplemented with Benzonase® (Merck). After cell disruption bysonication and clarification by centrifugation, each supernatant wasapplied on a gravity flow column with 1 ml agarose immobilized with ananti-ABD ligand (produced in-house). After washing with TST-buffer and 5mM NH₄Ac pH 5.5 buffer, the ABD fused Z variants were eluted with 0.1 MHAc. To fractions eluted in the anti-ABD agarose affinity chromatographypurification step, acetonitrile (ACN) was added to a final concentrationof 10% and the sample was loaded on 1 ml Resource 15RPC column (GEHealthcare), previously equilibrated with RPC solvent A (0.1% TFA, 10%ACN, 90% water). After column wash with RPC solvent A, bound proteinswere eluted with a linear gradient 0-50% RPC solvent B (0.1% TFA, 80%ACN, 20% water) for 20 ml. Fractions containing pure ABD-fused Zvariants were identified by SDS-PAGE analysis and pooled. After the RPCpurification, the buffer of the pool was exchanged to PBS (2.68 mM KCl,137 mM NaCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) using PD-10 columns(GE Healthcare). Finally, the ABD fused Z variants were purified on 1 mlEndoTrap® red columns (Hyglos) to ensure low endotoxin content.

Protein concentrations were determined by measuring the absorbance at280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinctioncoefficient of the respective protein. The purity was analyzed bySDS-PAGE stained with Coomassie Blue and the identity of each purified Zvariant was confirmed using LC/MS analysis.

Results

Cultivation and purification: The IL-17A binding Z variants with aHis₆-tag as well as the Z variants fused at their C-terminus to ABD,were expressed as soluble gene products in E. coli. The amount ofpurified protein from approximately 1-5 g bacterial pellet wasdetermined spectrophotometrically by measuring the absorbance at 280 nmand ranged from approximately 5 mg to 20 mg for the different IL-17Abinding Z variants. SDS-PAGE analysis of each final protein preparationshowed that these predominantly contained the IL-17A binding Z variant.The correct identity and molecular weight of each Z variant wereconfirmed by HPLC-MS analysis.

Example 3 Assessment of Blocking Ability of Primary IL-17A Binding ZVariants

Normal human dermal fibroblasts (NHDF) produce IL-6 upon stimulationwith IL-17A (Chang and Dong 2007, Cell Research 17:435-440) and theamount of released IL-6 to the supernatant is correlated to theconcentration of added IL-17A. Thus, blocking of IL-17A leads to areduction of IL-6 in the supernatant that can be quantified. In thisExample, the assay was used to evaluate the blocking ability of IL-17Abinding Z variants Z06260 (SEQ ID NO:1200), Z06282 (SEQ ID NO:1206) andZ06455 (SEQ ID NO:1214), alone or in fusion with ABD (PP013, SEQ IDNO:1224).

Materials and Methods

NHDF assay with His₆-tagged Z-variants: NHDF cells (Lonza, cat. no.CC-2511) were cultured in fibroblast basal medium (Lonza, cat. no.CC-3132) supplied with growth promoting factors (Lonza, cat. no.CC-5034). On the day before the experiment, 10⁵ cells were seeded in 100μl per well into 96-well culture plates (Greiner, cat. no. 655180). Onthe day of the experiment, dilutions of the IL-17A specific Z variantsHis₆-Z06260, His₆-Z06282 and His₆-Z06455 were prepared in a separate96-well plate. The Z variants were titrated in three-fold steps from3130 nM to 0.2 nM in medium containing 0.031 nM hIL-17A. A standardcurve of hIL-17A was also prepared (0.002-31 nM) as well as controlscontaining medium with 0.031 nM hIL-17A or medium alone. The medium inthe plate with the overnight cultured NHDF cells was discarded. Testsamples, standard curve samples and controls were transferred to thecell-containing plate. The NHDF cells were stimulated for 18-24 h at 37°C. and the IL-6 content in the supernatants was subsequently quantifiedusing the IL-6 specific ELISA described below.

NHDF assay with His₆-tagged and ABD fused Z variants: NHDF cells wereprepared as described above. On the day of the experiment, dilutions ofthe IL-17A specific Z variant constructs His₆-Z06282, His₆-Z06455,Z06260-ABD, Z06282-ABD and Z06455-ABD were prepared in a separate96-well plate. The Z variant constructs were titrated in four-fold stepsfrom 780 nM to 0.2 nM in medium containing 0.031 nM IL-17A and 8 pM HSA.Preparation of a standard IL-17A curve and controls, as well asdownstream analysis, was performed as described above.

IL-6 ELISA: 96-well half area plates (Costar, cat. no. 3690) were coatedwith the anti-IL-6 monoclonal antibody MAB206 (R&D systems) at aconcentration of 4 μg/ml in PBS (50 μl/well) and incubated overnight at4° C. On the next day, the plates were rinsed twice in tap water andblocked with PBS+2% BSA (Sigma) for 2 h. An IL-6 standard (R&D systems,cat. no. 206-IL-50), titrated in a 2-fold dilution series (0.2-50 ng/ml)and supernatants from the cell assay plate were added to the coatedELISA plates (50 μl/well) and incubated for 1.5 h at RT. The plates werewashed 4 times in an automated ELISA washer and 0.25 μg/ml (50 μl/well)of biotinylated anti-IL-6 polyclonal antibody (R&D systems, BAF206) wasadded. After incubation for 1 h, the plate was washed and 50 μl ofstreptavidin-HRP (Thermo Fisher, cat. no. N100) diluted 8000 times wasadded to each well. After one additional hour of incubation andsubsequent washing, the plate was developed with 50 μl TMB (ThermoFisher, cat. no. 34021) per well and the reactions were stopped with 50μl 2M H₂SO₄. The absorbance at 450 nm was measured in a 96-well platereader (Victor³) and the concentration of IL-6 in each sample wascalculated. The results are presented as the percentage of maximum IL-6production, calculated as:

100−[(ABS_(IL-17A blocker)−ABS_(background))/(ABS_(Max IL-6 prod)−ABS_(background))]×100

Results

Results from the first NHDF assay showed that all three Z variants

His₆-Z06260, His₆-Z06282 and His₆-Z06455 blocked IL-17A induced IL-6production in a dose dependent manner (FIG. 2A). The IC50 values wereapproximately 40-140 nM.

The NHDF assay was repeated with the same Z variants fused to ABD aswell as inclusion of HSA in the assay to ascertain retained binding tothe antigen when mimicking in vivo conditions. In vivo, ABD will bind toalbumin and convey longer circulation half-life. The results showed thatall three binders in fusion with ABD blocked IL-17A induced IL-6production equally well or better than the His₆-tagged Z variants (FIG.2B). The IC50 values from the second assay are summarized in Table 3.

TABLE 3 IC50 values for primary Z-variants blocking IL-17A induced IL-6production in the presence of HSA Analyte SEQ ID NO of Z variant IC50(nM) Z06260-ABD 1200 8 His₆-Z06282 1206 16 Z06282-ABD 1206 15His₆-Z06455 1214 67 Z06455-ABD 1214 15

Example 4 Design and Construction of a First Maturated Library of IL-17ABinding Z Variants

In this Example, a maturated library was designed and constructed. Thelibrary was used for selection of further IL-17A binding Z variants.Selections from maturated libraries are usually expected to result inbinders with increased affinity (Orlova et al., (2006) Cancer Res66(8):4339-48). In this study, randomized single stranded linkers weregenerated using split-pool DNA synthesis, enabling incorporation ofdefined codons in desired positions in the synthesis.

Materials and Methods

Library design: The library was based on the sequences of the IL-17Abinding Z variants identified as described in Example 1. In the newlibrary, 13 variable positions in the Z molecule scaffold were biasedtowards certain amino acid residues, according to a strategy based onthe Z variant sequences defined in SEQ ID NO:1200-1216. A DNA linker wasgenerated using split-pool synthesis containing the following 147 bpsequence ordered from DNA 2.0 (Menlo Park, Calif., USA): 5′-AA ATA AATCTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATCNNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTANNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO:1248, randomizedcodons are denoted NNN), comprising a coding sequence for a partiallyrandomized helix 1 and 2 in the corresponding amino acid sequence,flanked by restriction sites for XhoI and SacI. The theoreticaldistributions of amino acid residues in the new library including 13variable Z positions (9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and35) in the Z molecule scaffold are given in Table 4. The resultingtheoretical library size is 6.1×10⁹ variants.

TABLE 4 Library design, first maturation Amino acid position inRandomization No of the Z variant (amino acid amino moleculeabbreviations) acids Proportion 9 A, H, M, W, Y 5 ⅕ 10 A, D (60%), G, L,N 5 1/10, 6/10 (D) 11 D, E, F, N, Q R, Y 7 1/7 13 A, E, Q, W 4 ¼ 14 F,I, L, M, V, W 6 ⅙ 17 A, F, Q, W 4 ¼ 18 A, D, E, L, M, S, T 7 1/7 24 A,H, R, T, W, Y 6 ⅙ 25 A, D, H, R, V 5 ⅕ 27 A, G, Q, R, S, W 6 ⅙ 28 F, H,K, N, R, S, T, V, Y 9 1/9 32 A, G, H, I, Q, R, S, V 8 ⅛ 35 I, L, N, R 4¼

Library construction: The library was amplified using AmpliTaq Goldpolymerase (Applied Biosystems, cat. no. 4311816) during 12 cycles ofPCR, and pooled products were purified with QIAquick PCR PurificationKit (QIAGEN, cat. no. 28106) according to the supplier'srecommendations. The purified pool of randomized library fragments wasdigested with restriction enzymes XhoI and SacI-HF (New England Biolabs,cat. no. R0146L and cat. no. R3156M, respectively) and concentratedusing the QIAquick PCR Purification Kit. Subsequently, the product wassubjected to gel electrophoresis using a preparative 2.5% agarose gel(Nuisieve GTC agarose, Cambrex, Invitrogen) and purified from said gelusing QIAGEN Gel Extraction Kit (QIAGEN, cat. no. 28706) according tothe supplier's recommendations.

The phagemid vector pAY02592 (essentially as pAffi1 described inGrönwall et al., supra) was restricted with the same enzymes, purifiedusing phenol/chloroform extraction and ethanol precipitation. Therestricted fragments and the restricted vector were ligated in a molarratio of 5:1 with T4 DNA ligase (Fermentas, cat. no. EL0011) for 2 h atRT, followed by overnight incubation at 4° C. The ligated DNA wasrecovered by phenol/chloroform extraction and ethanol precipitation,followed by dissolution in 10 mM Tris-HCl, pH 8.5. Thus, the resultinglibrary in vector pAY02592 encoded Z variants each fused to an albuminbinding domain (ABD) derived from streptococcal Protein G.

The ligation reactions (approximately 160 ng DNA/transformation) wereelectroporated into electrocompetent E. coli ER2738 cells (Lucigen,Middleton, Wis., USA, 50 μl). Immediately after electroporation,approximately 1 ml of recovery medium (supplied with E. coli ER2738cells) was added. The transformed cells were incubated at 37° C. for 60min. Samples were taken for titration and for determination of thenumber of transformants. The cells were thereafter pooled and cultivatedovernight at 37° C. in 1 l of TSB-YE medium, supplemented with 2%glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. The cells werepelleted for 15 min at 4,000 g and resuspended in a PBS/glycerolsolution (approximately 40% glycerol). The cells were aliquoted andstored at −80° C. Clones from the library of Z variants were sequencedin order to verify the content and to evaluate the outcome of theconstructed library vis-à-vis the library design. Sequencing wasperformed as described in Example 1 and the amino acid distribution wasverified.

Preparation of phage stock: Phage stock containing the phagemid librarywas prepared in a 20 l fermenter (Belach Bioteknik). Cells from aglycerol stock containing the phagemid library were inoculated in 10 lof TSB-YE supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10mg/l tetracycline. When the cells reached an optical density at 600 nm(OD600) of 0.52, approximately 1.71 of the cultivation was infectedusing a 5× molar excess of M13K07 helper phage. The cells were incubatedfor 30 min, whereupon the fermenter was filled up to 10 l with complexfermentation medium [2.5 g/l (NH₄)₂SO₄, 5.0 g/l yeast extract; 30 g/ltryptone, 2 g/l K₂HPO₄; 3 g/l KH₂PO₄, 1.25 g/l, Na₃C₆H₅O₇.2 H₂O; BreoxFMT30 antifoaming agent 0.1 ml/1]. The following components were added:10 ml carbenicillin 25 mg/ml, 5 ml kanamycin 50 mg/ml, 1 ml 1 M IPTG;17.5 ml/I 1.217 M MgSO₄ and 5 ml of a trace element solution [129 mMFeCl₃; 36.7 mM ZnSO₄; 10.6 mM CuSO₄; 78.1 mM MnSO₄; 94.1 mM CaCl₂,dissolved in 1.2 M HCl]. A glucose limited fed-batch cultivation wasstarted where a 600 g/l glucose solution was fed to the reactor (3.5 g/hat the start, 37.5 g/h after 20 h continuing until the end of thecultivation). Through the automatic addition of 25% NH₄OH, the pH wascontrolled at pH 7. Air was supplemented (5 l/min) and the stirrer wasset at 500 rpm. After 24 h of fed-batch cultivation the OD₆₀₀ was 19.4.The cells in the cultivation were pelleted by centrifugation at 15,900g. The phage particles were precipitated from the supernatant twice inPEG/NaCl, filtered and dissolved in PBS and glycerol as described inExample 1. Phage stocks were stored at −80° C. until use in selection.

Results

Library construction: The new library was designed based on a set ofIL-17A binding Z variants with verified binding properties (Example 1and 3). The theoretical size of the designed library was 6.1×10⁹Zvariants. The actual size of the library, determined by titration aftertransformation to E. coli ER2738 cells, was 4.5×10⁹ transformants.

The library quality was tested by sequencing of 96 transformants and bycomparing their actual sequences with the theoretical design. Thecontents of the actual library compared to the designed library wereshown to be satisfactory. A maturated library of potential binders toIL-17A was thus successfully constructed.

Example 5 Selection, Screening and Characterization of Z Variants fromthe First Maturated Library Materials and Methods

Phage display selection of IL-17A binding Z variants: The targetproteins hIL-17A and mIL-17A were biotinylated as described inExample 1. Phage display selections, using the new library of Z variantmolecules constructed as described in Example 4, were performed in fourcycles against hIL-17A and mIL-17A essentially as described in Example1, with the following exceptions. Exception 1: PBST 0.1% was used asselection buffer. At selection, fetal calf serum (FCS, Gibco, cat. no.10108-165) and human serum albumin (HSA, Albucult, Novozymes, cat. no.230-005) were added to the selection buffer to a final concentration of10% and 1.5 μM, respectively. Exception 2: a pre-selection step wasperformed in cycle 1 by incubating the phage stock with SA beads.Exception 3: all tubes and beads used in the selection were pre-blockedwith PBS supplemented with 3% BSA and 0.1% Tween20. Exception 4: thetime for selection was 140, 70, 60 and 50 min in cycles 1, 2, 3 and 4,respectively.

An overview of the selection strategy, describing an increasedstringency in subsequent cycles obtained by using a lowered targetconcentration and an increased number of washes, is shown in Table 5.

TABLE 5 Overview of the selection from the first maturated library Phagestock Target Number Number Number of Selection from library or conc. of1 min of 10 min overnight Elution Cycle track selection track Target(nM) washes washes washes at 1 1 Zlib006IL- hIL-17A 25 2 — — pH 5.517A.I 1 2 Zlib006IL- hIL-17A 12.5 3 — — pH 2.2 17A.I 1 3 Zlib006IL-hIL-17A 25 2 — — pH 2.2 17A.I 2 1-1 1 hIL-17A 12.5 6 — — pH 5.5 2 2-1 2hIL-17A 2.5 10 — — pH 2.2 2 2-2 2 hIL-17A 5 6 — — pH 2.2 2 3-1 3 hIL-17A7.5 10 — — pH 2.2 2 3-2 3 hIL-17A 12.5 6 — — pH 2.2 2 3-3 3 mIL-17A 12.56 — — pH 2.2 3 1-1-1 1-1 hIL-17A 2.5 10 — — pH 5.5 3 2-1-1 2-1 hIL-17A0.05 15 1 — pH 2.2 3 2-2-1 2-2 hIL-17A 0.5 15 1 — pH 2.2 3 3-1-1 3-1hIL-17A 0.75 15 1 — pH 2.2 3 3-2-1 3-2 hIL-17A 2.5 10 — — pH 2.2 3 3-3-13-3 hIL-17A 5 10 — — pH 2.2 4 1-1-1-1a 1-1-1 hIL-17A 0.05 20 — — pH 5.54 1-1-1-1b 1-1-1 hIL-17A 0.05 20 — 1 pH 5.5 4 1-1-1-2a 1-1-1 hIL-17A 112 — — pH 5.5 4 1-1-1-2b 1-1-1 hIL-17A 1 12 — 1 pH 5.5 4 2-1-1-1a 2-1-1hIL-17A 0.0025 30 — — pH 2.2 4 2-1-1-1b 2-1-1 hIL-17A 0.0025 30 — 1 pH2.2 4 2-1-1-2a 2-1-1 hIL-17A 0.025 20 — — pH 2.2 4 2-1-1-2b 2-1-1hIL-17A 0.025 20 — 1 pH 2.2 4 2-2-1-1a 2-2-1 hIL-17A 0.005 30 — — pH 2.24 2-2-1-1b 2-2-1 hIL-17A 0.005 30 — 1 pH 2.2 4 2-2-1-2a 2-2-1 hIL-17A0.05 20 — — pH 2.2 4 2-2-1-2b 2-2-1 hIL-17A 0.05 20 — 1 pH 2.2 43-1-1-1a 3-1-1 hIL-17A 0.025 30 — — pH 2.2 4 3-1-1-1b 3-1-1 hIL-17A0.025 30 — 1 pH 2.2 4 3-1-1-2a 3-1-1 hIL-17A 0.25 20 — — pH 2.2 43-1-1-2b 3-1-1 hIL-17A 0.25 20 — 1 pH 2.2 4 3-2-1-1a 3-2-1 hIL-17A 0.0520 — — pH 2.2 4 3-2-1-1b 3-2-1 hIL-17A 0.05 20 — 1 pH 2.2 4 3-2-1-2a3-2-1 hIL-17A 1 12 — — pH 2.2 4 3-2-1-2b 3-2-1 hIL-17A 1 12 — 1 pH 2.2 43-3-1-1a 3-3-1 mIL-17A 0.05 20 — — pH 2.2 4 3-3-1-1b 3-3-1 mIL-17A 0.0520 — 1 pH 2.2 4 3-3-1-2a 3-3-1 mIL-17A 1 15 — — pH 2.2 4 3-3-1-2b 3-3-1mIL-17A 1 15 — 1 pH 2.2

Tracks 1-3 in cycle 1 were split in the second to fourth cycles,resulting in a total of six tracks (1-1 to 3-3) in cycle 2, six tracks(1-1-1 to 3-3-1) in cycle 3 and 24 tracks (1-1-1-la to 3-3-1-2b) incycle 4. Washes were performed using PBST 0.1% for 1 min. However, fortrack 1-3 in cycle 3, one of the washes lasted for 10 min.

After the last wash in cycle 4, the target-phage complexes on SA beadsfrom all tracks were divided in two equal parts. Bound phage particlesfrom the first part were immediately eluted, while the second part wassubjected to an overnight wash before elution of phage particles. Thebound phage particles were eluted using two different procedures: 1)with glycine-HCl, pH 2.2, as in Example 1, or 2) 500 μl of 100 mM sodiumphosphate, 150 mM sodium chloride, pH 5.5 and neutralization with 500 μlPBS.

Amplification of phage particles: Amplification of phage particlesbetween selection cycle 1 and 2 was performed essentially as describedin Example 1, with the following three exceptions. Exception 1: E. coliER2738 was used for phage amplification. Exception 2: M13K07 helperphage were used in 5× excess. Exception 3: the amplification of phageparticles between the selection cycles 2 and 4 was performed as follows:after infection of log phase E. coli ER2738 with phage particles, TSBsupplemented with 2% glucose, 10 μg/ml tetracycline and 100 μg/mlampicillin was added and followed by incubation with rotation for 30 minat 37° C. Next, the bacteria were infected with M13K07 helper phage. Theinfected bacteria were pelleted by centrifugation, re-suspended inTSB-YE medium supplemented with 100 μM IPTG, 25 μg/ml kanamycin and 100μg/ml ampicillin, and grown overnight at 30° C. The overnight cultureswere centrifuged, and phage particles in the supernatant wereprecipitated twice with PEG/NaCl buffer. Lastly, phages werere-suspended in selection buffer before entering the next selectioncycle.

In the final selection cycle, log phase bacteria were infected witheluate and diluted before spreading onto TBAB plates (30 g/l tryptoseblood agar base, Oxoid cat. no. CM0233B) supplemented with 0.2 g/lampicillin in order to form single colonies to be used in ELISAscreening.

Sequencing of potential binders: Individual clones from the differentselection tracks were picked for sequencing. Amplification of genefragments and sequence analysis of gene fragments were performedessentially as described in Example 1.

ELISA screening of Z variants: Single colonies containing Z variants(expressed as Z variant ABD fusion proteins as described in Example 1)were randomly picked from the selected clones of the maturated library,and grown in cultivations essentially as described in Example 1.Preparation of the periplasmic supernatants and ELISA screenings werealso performed essentially as described in Example 1 with the followingtwo exceptions. Exception 1: biotinylated hIL-17A was used at aconcentration of 0.4 nM. Exception 2: the periplasmic fraction of Zvariant Z06282 (SEQ ID NO:1206) from the primary selection was used as apositive control.

EC50 analysis of Z variants: A selection of IL-17A binding Z variantswas subjected to an analysis of response against a dilution series ofb-hIL-17A using ELISA as described above. Biotinylated target proteinwas added at a concentration of 6 nM and diluted stepwise 1:3 down to 8μM. As a background control, the Z variants were also assayed with notarget protein added. Periplasm samples containing the primary IL-17Abinder Z06282 (SEQ ID NO:1206) were included as a positive control. Datawere analyzed using GraphPad Prism 5 and non-linear regression, and EC50values (the half maximal effective concentration) were calculated.

Results

Phage display selection of maturated IL-17A binding Z variants:Selection was performed in a total of 24 parallel tracks containing fourcycles each. The different selection tracks differed in targetconcentration, target species origin (human IL-17A or murine IL-17A),selection time, wash conditions and the pH of the elution buffer. Clonesoriginating from the selection tracks using only human IL-17 and elutionat pH 2.2 were shown to have the best performance in ELISA screen.

Sequencing: Randomly picked clones were sequenced. Each individual Zvariant was given an identification number, Z #####, as described inExample 1. In total, 932 new unique Z variant molecules were identified.For the 494 best performing variants in the ELISA screen below, theamino acid sequences of the 58 amino acid residues long Z variants arelisted in FIG. 1 as SEQ ID NO:1-3, SEQ ID NO:11-16, SEQ ID NO:28-31, SEQID NO:36-63 and SEQ ID NO:67-519. The deduced IL-17A binding motifsextend from residue 8 to residue 36 in each sequence. The amino acidsequences of the 49 amino acid residues long polypeptides predicted toconstitute the complete three-helix bundle within each of these Zvariants extend from residue 7 to residue 55.

ELISA screening of Z variants: Clones obtained after four selectioncycles were produced in 96-well plates and screened for human IL-17Abinding activity using ELISA. All randomly picked clones were analyzed.494 of the 932 unique Z variants were found to give a response of 2× theblank control or higher (0.15-2.0 AU) against hIL-17A at a concentrationof 0.4 nM. Positive signals were shown for clones originating from allselection tracks. The blank controls had absorbances of 0.055-0.075 AU.

EC50 analysis of Z variants: A subset of Z variants was selected basedon the result of the ELISA screening experiment described above(absorbance over 1.25 AU) or based on variation in amino acid sequence,and subjected to a target titration in ELISA format. Periplasm sampleswere incubated with a serial dilution of b-hIL-17A ranging from 6 nM to8 μM. A periplasm sample containing Z06282 (SEQ ID NO:1206) identifiedin the primary selection was included as a positive control. Obtainedvalues were analyzed and the respective EC50 values were calculated(Table 6).

TABLE 6 Calculated EC50 values from ELISA titration analysis SEQ EC50 Zvariant ID NO (M) Z10210 36 1.1 × 10⁻⁹  Z10241 11 3.4 × 10⁻¹⁰ Z10255 374.6 × 10⁻¹⁰ Z10257 38 6.7 × 10⁻¹⁰ Z10433 28 9.4 × 10⁻¹⁰ Z10459 39 4.7 ×10⁻¹⁰ Z10462 12 3.4 × 10⁻¹⁰ Z10465 40 5.0 × 10⁻¹⁰ Z10470 41 4.7 × 10⁻¹⁰Z10483 42 4.0 × 10⁻¹⁰ Z10508 2 3.8 × 10⁻¹⁰ Z10529 43 6.4 × 10⁻¹⁰ Z105321 3.6 × 10⁻¹⁰ Z10534 13 4.1 × 10⁻¹⁰ Z10550 44 7.9 × 10⁻¹⁰ Z10565 45 3.8× 10⁻¹⁰ Z10566 14 5.5 × 10⁻¹⁰ Z10675 15 3.6 × 10⁻¹⁰ Z10676 46 4.0 ×10⁻¹⁰ Z10690 47 5.9 × 10⁻¹⁰ Z10703 48 7.5 × 10⁻¹⁰ Z10708 49 4.1 × 10⁻¹⁰Z10710 50 5.4 × 10⁻¹⁰ Z10718 16 5.1 × 10⁻¹⁰ Z10728 51 5.3 × 10⁻¹⁰ Z1074552 6.8 × 10⁻¹⁰ Z10756 53 6.4 × 10⁻¹⁰ Z10759 54 6.7 × 10⁻¹⁰ Z10775 55 5.1× 10⁻¹⁰ Z10778 56 4.7 × 10⁻¹⁰ Z10779 57 5.7 × 10⁻¹⁰ Z10800 58 6.4 ×10⁻¹⁰ Z10807 59 6.0 × 10⁻¹⁰ Z10844 60 7.5 × 10⁻¹⁰ Z10857 61 4.5 × 10⁻¹⁰Z10858 62 5.2 × 10⁻¹⁰ Z10859 31 6.9 × 10⁻¹⁰ Z10863 3 4.5 × 10⁻¹⁰ Z1091463 5.1 × 10⁻¹⁰ Z06282 1206 5.0 × 10⁻⁹ 

Example 6 Design and Construction of a Second Maturated Library ofIL-17A Binding Z Variants

In this Example, a second maturated library was constructed essentiallyas described in Example 4. The library was used for selections ofadditional IL-17A binding Z variants.

Materials and Methods

Library design: The library was based on the sequences of IL-17A bindingZ variants selected and characterized in Example 5. In the new library,the 13 positions in the Z molecule scaffold that were varied in thematuration library described in Examples 4 and 5 were biased towardscertain amino acid residues, according to a strategy based on the Zvariant sequences of the 37 top performing variants in the EC50 analysis(Table 6). A DNA linker was generated using split-pool synthesis andordered from DNA 2.0. It contained the following 147 bp, encodingpartially randomized helix 1 and 2 of the amino acid sequence: 5′-AA ATAAAT CTC GAG GTA GAT GCC/GCA AAA TAC GCC AAA GAA/GAG NNN NNN NNN GCG NNNNNN GAG ATC/ATT NNN NNN TTA/CTG CCT/CCC AAC TTA/CTC ACC NNN NNN CAA/CAGNNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTTA-3′ (SEQ ID NO:1249, randomized codons are denoted NNN) flanked byrestriction sites XhoI and SacI. The theoretical distributions of aminoacid residues in the new library, including six variable amino acidpositions (11, 14, 18, 25, 28 and 32) and seven constant amino acidpositions (9, 10, 13, 17, 24, 27 and 35) in the Z molecule scaffold aregiven in Table 7. The resulting theoretical library size is 3.9×10⁶variants.

Library construction: The library was constructed essentially asdescribed in Example 4 with the following exception: the cells werecultivated overnight in 0.5 I of TSB-YE medium, supplemented with 2%glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin.

Preparation of phage stock: Phage stock containing the phagemid librarywas prepared in shake flasks. Cells from a glycerol stock containing thephagemid library were inoculated in 0.51 of TSB-YE medium, supplementedwith 2% glucose, 10 μg/ml tetracycline and 100 μg/ml ampicillin. Thecultivations were grown at 37° C. until OD₆₀₀ reached 0.6 and thenapproximately 83 ml of the cultivation was infected using a 5× molarexcess of M13K07 helper phage and incubated for 40 min at 37° C. Thecells in the cultivation were pelleted by centrifugation, dissolved inTSB-YE medium, supplemented with 100 μg/ml ampicillin, 25 μg/mlkanamycin and 0.1 mM IPTG and grown at 30° C. for 18 h. Aftercultivation, the cells were pelleted by centrifugation at 4,000 g andthe phage particles remaining in the medium were thereafter precipitatedtwice in PEG/NaCl, filtered and dissolved in PBS and glycerol asdescribed in Example 1. Phage stocks were stored at −80° C. until use inselection.

TABLE 7 Library design, second maturation Amino acid position inRandomization No of the Z variant (amino acid amino moleculeabbreviations) acids Proportion 9 A 1 1/1 10 D 1 1/1 11 A, D, E, F, G,H, I, K, L, M, 17 1/17 N, Q, R, S, T V, W 13 A 1 1/1 14 A, F, I, L, M,V, Y 7 1/7 17 A 1 1/1 18 A, D, E, F, G, H, I, K, L, M, 17 1/17 N, Q, R,S, V, W, Y 24 W 1 1/1 25 A, D (50%), E, F, I, L, M, V, 10 1/18, W, Y ½(0) 27 W 1 1/1 28 A, D, E, F, G, H, I, L, M, Q, 12 1/12 W, Y 32 A, D, E,F, G, H, K, L, M, N, 16 1/16 Q, R, S, T, W, Y 35 R 1 1/1

Results

Library construction: The new library was designed based on a set ofIL-17A binding Z variants with verified binding properties (Example 5).The theoretical size of the designed library was 3.9×10⁶ Z variants. Theactual size of the library, determined by titration after transformationto E. coli. ER2738 cells, was 1.4×10⁹ transformants.

The library quality was tested by sequencing of 96 transformants and bycomparing their actual sequences with the theoretical design. Thecontents of the actual library compared to the designed library wereshown to be satisfactory. A maturated library of potential binders toIL-17A was thus successfully constructed.

Example 7 Selection, Screening and Characterization of Z Variants fromthe Second Maturated Library Materials and Methods

Phage display selection of IL-17A binding Z variants: The target proteinhIL-17A was biotinylated as described in Example 1. Phage displayselections, using the second maturated library of Z variant moleculesconstructed as described in Example 6, were performed against hIL-17Aessentially as described in Example 5 with the following exceptions.Exception 1: selections were performed in solution or at solid phase inRT. Exception 2: the time for selection was 60 min, 10 min or 1 min incycle 1 and 30 min, 4 min or 10 sec in cycles 2, 3 and 4. Selection insolution was followed by catching of target-phage complexes on SA beadsas described in Example 1. Exception 3: during selection on solid phase,the target was caught on SA beads prior to selection.

An overview of the selection strategy, summarizing the differencesbetween selection in solution and selection on solid phase, as well asthe increased stringency in subsequent cycles obtained by using alowered target concentration and an increased number of washes, is shownin Table 8.

Tracks 1-6 in cycle 1 were divided in the second to fourth cycles,resulting in total of seven tracks (1-1 to 6-1) in cycle 2, eight tracks(1-1-1 to 6-1-1) in cycle 3 and 16 tracks (1-1-1-1 to 6-1-1-1) in cycle4. Washes were performed using PBST 0.1% during 1 min. However, one ofthe washes lasted for 15 min in tracks 2-1-1, 3-1-1 and 5-1-1 in cycle3.

After the last wash in cycle 4, the target-phage complexes on SA beadsfrom five of the tracks (1-1-1-1, 1-2-1-2, 3-1-1-1, 4-1-1-1 and 5-1-1-2)were divided in two equal parts. Bound phage particles from the firstpart were immediately eluted, while the second part was subjected to awash during approximately 65 h before the elution of phage particles.The bound phage were eluted using glycine-HCl, pH 2.2, as described inExample 1.

The amplification of phage particles between selection cycles wasperformed essentially as described in Example 1.

TABLE 8 Overview of the selection from the second maturated libraryPhage stock Target Selection Number Number Number Selection from libraryor conc. time of 1 min of 15 min of ~65 h Selection Cycle trackselection track (pM) (min) washes washes washes method 1 1 Zlib006IL-12500 60 3 — — solution 17A.II 1 2 Zlib006IL- 12500 60 3 — — solution17A.II 1 3 Zlib006IL- 1250 60 3 — — solution 17A.II 1 4 Zlib006IL- 1250010 3 — — solid 17A.II phase 1 5 Zlib006IL- 2500 10 3 — — solid 17A.IIphase 1 6 Zlib006IL- 2500 1 3 — — solid 17A.II phase 2 1-1 1 5000 30 6 —— solution 2 1-2 1 2500 30 10 — — solution 2 2-1 2 1000 30 10 — —solution 2 3-1 3 100 30 10 — — solution 2 4-1 4 5000 4 6 — — solid phase2 5-1 5 250 4 10 — — solid phase 2 6-1 6 250 0.17 10 — — solid phase 31-1-1 1-1 500 30 15 — — solution 3 1-2-1 1-2 50 30 15 — — solution 32-1-1 2-1 25 30 14 1 — solution 3 3-1-1 3-1 5 30 14 1 — solution 3 4-1-14-1 500 4 15 — — solid phase 3 4-1-2 4-1 50 4 15 — — solid phase 3 5-1-15-1 25 4 14 1 — solid phase 3 6-1-1 6-1 5 0.17 15 — — solid phase 41-1-1-1 1-1-1 50 30 20 — — solution 4 1-1-1-1X 1-1-1 50 30 20 — 1solution 4 1-1-1-2 1-1-1 5 30 20 — — solution 4 1-2-1-1 1-2-1 25 30 20 —— solution 4 1-2-1-2 1-2-1 5 30 20 — — solution 4 1-2-1-2X 1-2-1 5 30 20— 1 solution 4 2-1-1-1 2-1-1 0.5 30 20 — — solution 4 3-1-1-1 3-1-1 0.0530 20 — — solution 4 3-1-1-1X 3-1-1 0.05 30 20 — 1 solution 4 4-1-1-14-1-1 50 4 20 — — solid phase 4 4-1-1-1X 4-1-1 50 4 20 — 1 solid phase 44-1-2-1 4-1-2 25 4 20 — — solid phase 4 5-1-1-1 5-1-1 2.5 4 20 — solidphase 4 5-1-1-2 5-1-1 0.5 4 20 — — solid phase 4 5-1-1-2X 5-1-1 0.5 4 20— 1 solid phase 4 6-1-1-1 6-1-1 0.5 0.17 20 — — solid phase

Sequencing of potential binders: Individual clones from the differentselection tracks were picked for sequencing. Amplification and sequenceanalysis of gene fragments were performed essentially as described inExample 1.

ELISA screening of Z variants: Single colonies containing Z variants(expressed as Z variant ABD fusion proteins as described in Example 1)were randomly picked from the selected clones of the IL-17A secondmaturated library and grown in cultivations as described in Example 1.Preparation of the periplasmic supernatants and ELISA screenings wereperformed essentially as described in Example 1 with the following twoexceptions. Exception 1: biotinylated hIL-17A was used at aconcentration of 0.2 or 0.33 nM. Exception 2: the periplasmic fractionof Z variant Z10241 (SEQ ID NO:11), identified in selections from thefirst maturated library, was used as a positive control and the blankcontrol was created by exchanging the periplasmic step with addition ofPBST 0.05%.

EC50 analysis of Z variants: A selection of IL-17A binding Z variantswas subjected to an analysis of the response against a dilution seriesof b-hIL-17A using ELISA as described in Example 5. Biotinylated proteinwas added at a concentration of 10 nM and diluted stepwise 1:2 eighttimes followed by one 1:5 dilution down to 8 μM. As a backgroundcontrol, the Z variants were also assayed with no target protein added.A periplasm sample containing the previously maturated Z variant Z10241(SEQ ID NO:11) was included as positive control. Data were analyzedusing GraphPad Prism 5 and non-linear regression and EC50 values werecalculated.

Results

Phage display selection of IL-17A binding Z variants from a secondmaturated library: Selection was performed in a total of 16 paralleltracks containing four cycles each. The selection tracks differed intarget concentration, selection time, wash conditions and if theselection was performed in solution or on solid phase.

Sequencing: Randomly picked clones were sequenced. Each individual Zvariant was given an identification number, Z #####, as described inExample 1. In total, 759 new unique Z variant molecules were identified.

For the 704 best performing variants in the ELISA screen below, theamino acid sequences of the 58 amino acid residues long Z variants arelisted in FIG. 1 and in the sequence listing as SEQ ID NO:5-10, SEQ IDNO:17-27, SEQ ID NO:32-35, SEQ ID NO:64-66 and SEQ ID NO:520-1199. Thededuced IL-17A binding motifs extend from residue 8 to residue 36 ineach sequence. The amino acid sequences of the 49 amino acid residueslong polypeptides predicted to constitute the complete three-helixbundle within each of these Z variants extend from residue 7 to residue55.

ELISA screening of Z variants: Clones obtained after four selectioncycles were produced in 96-well plates and screened for hIL-17A bindingactivity using ELISA. All randomly picked clones were analyzed. 704 ofthe 759 unique Z variants were found to give a response of 2× the blankcontrols or higher (0.22-2.2 AU) against hIL-17A at a concentration of0.2 or 0.33 nM. Positive signals were collected for clones originatingfrom all selection tracks. The average response of the blank controlswas 0.11 AU, based on a representative set of plates.

EC50 analysis of Z variants: A subset of IL-17A binding Z variants wasselected based on the result in the ELISA experiment described above (Zvariants with absorbances over the response of the positive controlZ10241, SEQ ID NO:11) and subjected to a target titration in ELISAformat as described in Example 5. Obtained values were analyzed andtheir respective EC50 values were calculated (Table 9).

TABLE 9 Calculated EC50 values from ELISA titration analysis SEQ EC50 Zvariant ID NO (M) Z12059 17 5.3 × 10⁻¹⁰ Z12060 5 5.1 × 10⁻¹⁰ Z12073 184.3 × 10⁻¹⁰ Z12078 8 5.2 × 10⁻¹⁰ Z12081 6 5.6 × 10⁻¹⁰ Z12115 20 4.2 ×10⁻¹⁰ Z12163 9 5.0 × 10⁻¹⁰ Z12180 21 3.7 × 10⁻¹⁰ Z12211 22 4.6 × 10⁻¹⁰Z12212 64 7.8 × 10⁻¹⁰ Z12256 23 4.7 × 10⁻¹⁰ Z12264 10 3.9 × 10⁻¹⁰ Z1227524 4.2 × 10⁻¹⁰ Z12285 65 9.1 × 10⁻¹⁰ Z12439 66 6.1 × 10⁻¹⁰

Example 8 In Vitro Characterization of a Subset of Maturated IL-17ABinding Z Variants Materials and Methods

Subcloning and production of Z variants with an N-terminal His₆-tag wereperformed as described in Example 2. One additional variant, Hise-Z15167(SEQ ID NO:4), was created by site directed mutagenesis of Hise-Z10532(SEQ ID NO:1) resulting in substitution of Din position 25 with A.Production of His₆-Z15167 was performed as described above for other Zvariants.

Circular dichroism (CD) spectroscopy analysis: Purified His₆-tagged Zvariants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, aCD spectrum at 250-195 nm was obtained at 20° C. In addition, a variabletemperature measurement (VTM) was performed to determine the meltingtemperature (Tm). In the VTM, the absorbance was measured at 221 nmwhile the temperature was raised from 20 to 90° C., with a temperatureslope of 5° C./min. A new CD spectrum was obtained at 20° C. after theheating procedure in order to study the refolding ability of the Zvariants. The CD measurements were performed on a Jasco J-810spectropolarimeter (Jasco Scandinavia AB) using a cell with an opticalpath length of 1 mm.

IL-17 binding ELISA screening: 96-well half area plates (Costar, 3690)were coated overnight at 4° C. with hIL-17A at 1 μg/ml in PBS in avolume of 50 μl/well. On the day of analysis, the plate was rinsed twicein tap water and then blocked with PBS+2% BSA (Sigma) for 2 h. Z10241(SEQ ID NO:11) was used as a standard and was titrated in a 3-folddilution series (300-0.005 ng/ml) and the other Z variants were added infour different dilutions to the coated ELISA plate (50 μl/well) andincubated for 1.5 h at RT. The plate was washed 4 times in an automatedELISA washer and 2 μg/ml (50 μl/well) of a goat anti-Z antibody wasadded. After 1 h of incubation, the plate was washed and 50 μl ofanti-goat IgG-HRP (DAKO) diluted 5000 times was added per well. Theplate was developed after another 1 h incubation, washed with 50 μl TMB(Thermo Fisher, 34021) per well and the reaction was stopped with 50 μl2 M H2504. The plates were read in a multi label reader (Victor³, PerkinElmer).

Biacore kinetic analysis: Kinetic constants (k_(on) and k_(off)) andaffinities (K_(D)) for human IL-17A were determined for 27 His₆-tagged Zvariants. The hIL-17A was immobilized in the flow cell on thecarboxylated dextran layer of a CM5 chip surface (GE Healthcare, cat.no. BR100012). The immobilization was performed using amine couplingchemistry according to the manufacturer's protocol and using HBS-EP(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20,GE Healthcare, cat. no. BR100188) as running buffer. One flow cellsurface on the chip was activated and deactivated for use as blankduring analyte injections. In the kinetic experiment, HBS-EP was used asrunning buffer and the flow rate was 50 μl/min. The analytes, i.e. the Zvariants, were each diluted in HBS-EP buffer to final concentrations of100 nM, 20 nM and 4 nM and injected for 4 min, followed by dissociationin running buffer for 8 min. After 8 min dissociation, the surfaces wereregenerated with two injections of 10 mM HCl. Kinetic constants werecalculated from the sensorgrams using the Langmuir 1:1 model ofBiaEvaluation software 4.1 (GE Healthcare).

Biacore binding specificity analysis: The interactions of threeHise-tagged IL-17A binding Z variants (Z10508 (SEQ ID NO:2), Z10532 (SEQID NO:1) and Z15167 (SEQ ID NO:4)) with hIL-17A (SEQ ID NO:1226),hIL-17F (SEQ ID NO:1228, R&D Systems, cat. no. 1335-IL/CF) and the humanIL-17A/F heterodimer (R&D Systems, cat. no. 5194-IL-025/CF) wereanalyzed in a Biacore 2000 instrument. The three different IL-17variants were immobilized in the flow cells on the carboxylated dextranlayer of a CM5 chip surface. The immobilization was performed usingamine coupling chemistry according to the manufacturer's protocol andusing HBS-EP as running buffer. One flow cell surface on the chip wasactivated and deactivated for use as blank during analyte injections. Inthe binding experiment, HBS-EP was used as running buffer and the flowrate was 50 μl/min. The analytes, i.e. the Z variants, were each dilutedin HBS-EP running buffer to final concentrations of 4, 20, 100 and 500nM and injected for 4 min. After 15 min of dissociation, the surfaceswere regenerated with four injections of 10 mM HCl. The results wereanalyzed using the BiaEvaluation software.

Blocking of IL-17A induced IL-6 production in the NHDF assay: The NHDFassay and quantification by the IL-6 specific ELISA was performedessentially as described in Example 3. In brief, on the day before theexperiment, 5000 cells were seeded per well into a half area 96-wellculture plates in 100 μl. On the day of the experiment, dilutions of 36IL-17A specific Z variants were prepared in a separate 96-well plate.The Z variants were titrated in three-fold steps from 4690 nM to 0.08 nMin medium containing 0.9 nM hIL-17A. A standard curve of hIL-17A(6.2-0.0001 nM) was prepared, as well as controls containing medium with0.9 nM hIL-17A or medium alone.

Results

CD analysis: The CD spectra determined for the IL-17A binding Z variantswith a His₆ tag showed that each had a α-helical structure at 20° C. Theresults of the variable temperature measurements, wherein meltingtemperatures (Tm) were determined, are shown in Table 10. Reversiblefolding was seen for all the IL-17A binding Z variants when overlayingspectra measured before and after heating to 90° C.

Analysis of IL-17A binding capacity by ELISA: Purified His₆-tagged Zvariant molecules from the first and second maturation round werescreened for their capacity to bind IL-17A in an ELISA assay. Theresults are shown in FIG. 3 as percent binding capacity compared to thevariant Z10241.

Biacore kinetic analysis: The interactions of 28 His₆-taggedIL-17A-binding Z variants with hIL-17A were analyzed in a Biacoreinstrument by injecting various concentrations of the Z variants over asurface containing immobilized IL-17A. A summary of the kineticparameters (KD, ka (kon) and kd (koff)) for binding of the Z variants tohIL-17A using a 1:1 interaction model is given in Table 11.

TABLE 10 Melting temperatures (Tm) SEQ ID NO Analyte of Z variant Tm (°C.) His6-Z10241 11 48 His6-Z10433 28 56 His6-Z10462 12 51 His6-Z10508 254 His6-Z10532 1 57 His6-Z10534 13 41 His6-Z10566 14 51 His6-Z10675 1552 His6-Z10681 29 52 His6-Z10718 16 48 His6-Z10722 30 51 His6-Z10859 3152 His6-Z10863 3 51 His6-Z12059 17 50 His6-Z12060 5 52 His6-Z12073 18 49His6-Z12077 19 52 His6-Z12078 8 51 His6-Z12081 6 50 His6-Z12115 20 51His6-Z12163 9 54 His6-Z12180 21 51 His6-Z12192 32 45 His6-Z12211 22 49His6-Z12256 23 47 His6-Z12264 10 54 His6-Z12275 24 46 His6-Z12283 25 45His6-Z12289 33 51 His6-Z12344 26 49 His6-Z12481 27 49 His6-Z12498 34 48His6-Z12522 35 51 His6-Z12634 7 50 Z10241-His6 11 47 His6-Z15167 4 57Z10199 1217 56

Biacore binding specificity analysis: The binding of three His₆-taggedIL-17A binding Z variants (Z10508, Z10532 and Z15167) to hIL-17A,hIL-17F and hIL-17A/F were tested in a Biacore instrument by injectingthe Z variants over surfaces containing the IL-17 variants. The ligandimmobilization levels on the surfaces were 657 RU, 977 RU and 770 RU ofIL-17A, IL-17F and IL-17A/F, respectively. All tested Z variants showedbinding to human IL-17A and weaker binding to IL-17A/F, whereas nobinding to IL-17F could be detected. The resulting curves, from whichresponses from a blank surface were subtracted, are displayed in FIGS.4A-4C. Z15167 showed the fastest association curve to human IL-17A.

Blocking of IL-17A induced IL-6 production in the NHDF assay: PurifiedHis₆-tagged Z variants from the first and second maturation rounds werescreened for their capacity to block IL-17A induced IL-6 production inthe NHDF assay. Results from the NHDF assay showed that all testedmaturated binders had an increased IL-17A specific blocking capacitycompared to the primary binder His₆-Z06282. A graph displaying typicalinhibition profiles of a selection of binders from the first maturationlibrary are shown in FIG. 5, and the calculated IC50 values for allanalysed binders are shown in Table 12.

TABLE 11 Kinetic parameters for binding of Z variants to hIL-17A SEQ IDNO Analyte of Z variant k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) His6-Z1024111 5.2 × 10⁵ 1.2 × 10⁻³ 2.2 × 10⁻⁹ His6-Z10433 28 4.7 × 10⁵ 3.1 × 10⁻³6.4 × 10⁻⁹ His6-Z10508 2 5.1 × 10⁵ 2.0 × 10⁻³ 4.0 × 10⁻⁹ His6-Z10532 16.2 × 10⁵ 1.5 × 10⁻³ 2.4 × 10⁻⁹ His6-Z10566 14 6.1 × 10⁵ 5.7 × 10⁻³ 9.3× 10⁻⁹ His6-Z10675 15 5.7 × 10⁵ 1.1 × 10⁻³ 1.9 × 10⁻⁹ His6-Z10681 29 6.7× 10⁵ 3.5 × 10⁻³ 5.2 × 10⁻⁹ His6-Z10859 31 5.6 × 10⁵ 6.8 × 10⁻³ 1.2 ×10⁻⁸ His6-Z10863 3 8.3 × 10⁵ 2.5 × 10⁻³ 3.0 × 10⁻⁹ His6-Z12060 5 7.5 ×10⁵ 3.3 × 10⁻³ 4.3 × 10⁻⁹ His6-Z12073 18 4.9 × 10⁵ 1.2 × 10⁻³ 2.3 × 10⁻⁹His6-Z12077 19 6.5 × 10⁵ 2.0 × 10⁻³ 3.1 × 10⁻⁹ His6-Z12078 8 4.5 × 10⁵8.1 × 10⁻⁴ 1.8 × 10⁻⁹ His6-Z12081 6 6.2 × 10⁵ 3.8 × 10⁻³ 6.2 × 10⁻⁹His6-Z12163 9 7.3 × 10⁵ 1.6 × 10⁻³ 2.2 × 10⁻⁹ His6-Z12192 32 4.6 × 10⁵5.6 × 10⁻³ 1.2 × 10⁻⁸ His6-Z12211 22 5.8 × 10⁵ 1.4 × 10⁻³ 2.4 × 10⁻⁹His6-Z12256 23 3.5 × 10⁵ 1.2 × 10⁻³ 3.4 × 10⁻⁹ His6-Z12264 10 6.0 × 10⁵5.5 × 10⁻⁴  9.3 × 10⁻¹⁰ His6-Z12275 24 5.2 × 10⁵ 1.1 × 10⁻³ 2.0 × 10⁻⁹His6-Z12283 25 5.5 × 10⁵ 1.8 × 10⁻³ 3.3 × 10⁻⁹ His6-Z12289 33 6.2 × 10⁵3.3 × 10⁻³ 5.3 × 10⁻⁹ His6-Z12344 26 7.1 × 10⁵ 6.6 × 10⁻⁴  9.2 × 10⁻¹⁰His6-Z12481 27 5.2 × 10⁵ 8.9 × 10⁻⁴ 1.7 × 10⁻⁹ His6-Z12498 34 6.0 × 10⁵2.3 × 10⁻³ 3.8 × 10⁻⁹ His6-Z12522 35 7.1 × 10⁵ 4.4 × 10⁻³ 6.2 × 10⁻⁹His6-Z12634 7 6.9 × 10⁵ 4.3 × 10⁻³ 6.3 × 10⁻⁹ His6-Z15167 4 1.3 × 10⁶1.4 × 10⁻³ 1.1 × 10⁻⁹

TABLE 12 IC50 values for maturated Z-variants SEQ ID NO IC50 Analyte ofZ variant: (nM) His6-Z10241 11 5.3 His6-Z10433 28 14 His6-Z10462 12 6.6His6-Z10508 2 5.7 His6-Z10532 1 6.4 His6-Z10534 13 6.5 His6-Z10566 149.6 His6-Z10675 15 5.3 His6-Z10681 29 25 His6-Z10718 16 28 His6-Z1072230 35 His6-Z10859 31 26 His6-Z10863 3 8.5 His6-Z12059 17 3.7 His6-Z120605 7.9 His6-Z12073 18 4.0 His6-Z12077 19 5.5 His6-Z12078 8 4.5His6-Z12081 6 8.1 His6-Z12115 20 3.0 His6-Z12163 9 7.0 His6-Z12180 214.3 His6-Z12192 32 28 His6-Z12211 22 2.9 His6-Z12256 23 4.4 His6-Z1226410 4.4 His6-Z12275 24 4.8 His6-Z12283 25 6.9 His6-Z12289 33 18His6-Z12344 26 4.8 His6-Z12481 27 3.0 His6-Z12498 34 11 His6-Z12522 3510 His6-Z12634 7 9.8 Z10241-His6 11 3.3

Example 9 Production of IL-17 Binding Z-ABD-Z Polypeptides

IL-17A is a homodimeric cytokine that possesses two receptor bindingsites. It was speculated that a polypeptide comprising two moieties ofan IL-17A binding Z variant of the present disclosure would block IL-17Amore efficiently than a polypeptide comprising one such moiety. ThisExample describes the general procedure for subcloning and production ofpolypeptides comprising two Z variants in fusion with the albuminbinding domain variant PP013 (SEQ ID NO:1224) in the general formatZ-[L1]-ABD-[L2]-Z where [L1] and [L2] are linkers separating the Z andABD moieties.

Materials and Methods

Subcloning of Z-[L1]-ABD-[L2]-Z polypeptides with different [L1] and[L2] linker lengths: The DNA of Z06282 (SEQ ID NO:1206), Z10241 (SEQ IDNO:11) and Z10532 (SEQ ID NO:1) were amplified from the library vectorpAY02592 by PCR using Pfu Turbo DNA polymerase (Agilent Technologies,cat. no. 600254) together with suitable primer pairs. TheZ-[L1]-ABD-[L2]-Z constructs were generated by ligation of DNA encodingeach moiety into the expression vector pET26b(+) (Novagen, Madison,Wl)in three subsequent cloning steps using T4 DNA ligase (Fermentas, cat.no. EL0011). DNA encoding the three moieties were separated by DNAencoding hybridized linkers with different number of repeats of(GGGGS)_(n), flanked by restriction enzyme sites. The constructs encodedby the expression vectors were Z #####-[GAP-(G₄S[SEQ IDNO:1301])n-TS]-P013-[GT-(G₄S[SEQ ID NO:1301])_(n)-PR]-Z #####, whereeach n individually is 1-4, and as further specified in Table 13.

TABLE 13 Dimeric Z variants fused to ABD via different [L1] and [L2]lengths SEQ Designation ID NO Z-[L1]-ABD-[L2]-Z polypeptide ZAZ3174 1233Z06282-[GAP-(G₄S)₄-TS]-PP013-[GT-(G₄S)₄- PR]-Z06282 ZAZ3175 1234Z06282-[GAP-(G₄S)₃-TS]-PP013-[GT-(G₄S)₃- PR]-Z06282 ZAZ3176 1235Z06282-[GAP-(G₄S)₂-TS]-PP013-[GT-(G₄S)₂- PR]-Z06282 ZAZ3236 1240Z06282-[GAP-G₄S-TS]-PP013-[GT-G₄S- PR]-Z06282 ZAZ3237 1241Z06282-[GAP-G₄S-TS]-PP013-[GT-(G₄S)₂- PR]-Z06282 ZAZ3220 1236Z10241-[GAP-(G₄S)₄-TS]-PP013-[GT-(G₄S)₄- PR]-Z10241 ZAZ3221 1237Z10532-[GAP-(G₄S)₄-TS]-PP013-[GT-(G₄S)₄- PR]-Z10532Restriction sites (I)-(IV) in DNA encoding Z#####-[(I)-(G₄S)n-(II)]-PP013-[(III)-(G₄S)n-(IV)]-Z ##### are cleavablewith restriction enzymes AscI (I), SpeI (II), KpnI (III) and SacII (IV),respectively

Subcloning of Z-[L1]-ABD-[L2]-Z polypeptides with a minimal [L1] linker:DNA encoding additional dimeric Z variants comprising Z06282 (SEQ IDNO:1206) but with a minimal linker [L1] were generated using standardmolecular biology techniques. The constructs encoded by the expressionvectors were Z06282-VDGS (SEQ ID NO:1311)-PP013-GT-(G₄S[SEQ IDNO:1301])_(n)-PR-Z06282 and as further specified in Table 14.

The genes encoding Z12876 (SEQ ID NO:1218), Z14253 (SEQ ID NO:1219),Z14254 (SEQ ID NO:1220) and Z14255 (SEQ ID NO:1221) (corresponding toZ10241 (SEQ ID NO:11), Z10532 (SEQ ID NO:1), Z10508 (SEQ ID NO:2) andZ10863 (SEQ ID NO:3), respectively, but starting with the amino acidresidues AE instead of VD) were amplified by PCR using Pfu Turbo DNApolymerase together with suitable primer pairs. The Z-[L1]-ABD-[L2]-Zconstructs were generated by ligation of each fragment into theexpression vector pET26b(+) in three subsequent cloning steps using T4DNA ligase. DNA encoding the three moieties were separated by linkersfurther modified by site-directed mutagenesis using standard molecularbiology techniques. The constructs encoded by the expression vectorswere Z ####-[VDGS(SEQ ID NO:1311)]-PP013-[GT-G₄S-PK(SEq ID NO:1317)]-Z#### and Z #####-[ASGS(SEQ ID NO:1312)]-PP013-[GT-G₄S(SEQ ID NO:1306)]-Z#####, and as further specified in Table 14.

TABLE 14 Dimeric Z variants fused to ABD via a minimal [L1] linker SEQID Designation NO Z-[L1]-ABD-[L2]-Z polypeptide Mutations ZAZ3234 1238Z06282-[VDGS]-PP013- GT-G₄S-PR-Z06282 ZAZ3235 1239 Z06282-[VDGS]-PP013-GT-(G₄S)₂-PR-Z06282 ZAZ3269 1242 Z12876-[VDGS]-PP013- R117K[GT-G₄S-PK]-Z12876 ZAZ3270 1243 Z12876-[ASGS]-PP013- V59A, D60S,[GT-G₄S]-Z12876 Δ116P, Δ117R ZAZ3363 1244 Z14253-[ASGS]-PP013- V59A,D60S, [GT-G₄S]-Z14253 Δ116P, Δ117R ZAZ3364 1245 Z14254-[ASGS]-PP013-V59A, D60S, [GT-G₄S]-Z14254 Δ116P, Δ117R ZAZ3365 1246Z14255-[ASGS]-PP013- V59A, D60S, [GT-G₄S]-Z14255 Δ116P, Δ117R ZAZ34221247 Z15166-[ASGS]-PP013- D25A in both [GT-G₄S]-Z15166 Z14253 moietiesin ZAZ3363 Δdeletion of the indicated amino acid residue

Cultivation: E. coli BL21(DE3) cells (Novagen) were transformed withplasmids containing the gene fragment of each respective Z-ABD-Zpolypeptide and cultivated at 37° C. in 800 or 1000 ml of TSB-YE mediumsupplemented with 50 μg/ml kanamycin. In order to induce proteinexpression, IPTG was added to a final concentration of 0.2 mM at OD600=2and the cultivation was incubated at 37° C. for another 5 h. The cellswere harvested by centrifugation.

Purification of IL-17A binding Z-ABD-Z polypeptides: Cell pellets werere-suspended in TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl,0.05% Tween 20, pH 8.0) supplemented with Benzonase® (Merck). After celldisruption by sonication and clarification by centrifugation, eachsupernatant was applied onto a column packed with agarose andimmobilized with an anti-ABD ligand (produced in-house). After washingwith TST buffer and 5 mM NH₄Ac pH 5.5 buffer, the Z-ABD-Z polypeptideswere eluted with 0.1 M HAc. Acetonitrile (ACN) was added to elutedfractions to a final concentration of 10% and the samples were loaded onSOURCE 15RPC columns (GE Healthcare), previously equilibrated with RPCsolvent A (0.1% TFA, 10% ACN, 90% water). After column wash with RPCsolvent A, bound proteins were eluted with a linear gradient from RPCsolvent A to RPC solvent B (0.1% TFA, 80% ACN, 20% water). Fractionscontaining pure Z-ABD-Z polypeptides were identified by SDS-PAGEanalysis and pooled. After the RPC purification, the buffer of the poolwas exchanged to PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄, pH 7.4) using Sephadex G-25 columns (GE Healthcare). Finally,the Z-ABD-Z variants were purified on EndoTrap® red columns (Hyglos) toensure low endotoxin content.

Protein concentrations were determined by measuring the absorbance at280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinctioncoefficient of the respective protein. Purity was analyzed by SDS-PAGEstained with Coomassie Blue, and the identity of each purified Z-ABD-Zvariant was confirmed by HPLC-MS analysis.

Results

Cultivation and purification: The IL-17A binding Z variants in fusionwith ABD were expressed as soluble gene products in E. coli. SDS-PAGEanalysis of each final protein preparation showed that thesepredominantly contained the desired IL-17A binding Z-ABD-Z polypeptide.The correct identity and molecular weight of each Z-ABD-Z polypeptidewere confirmed by HPLC-MS analysis.

Example 10 Solubility of Z-ABD-Z Polypeptides

The solubility of three Z-ABD-Z polypeptides in physiological buffer wasinvestigated by consecutive concentrations of the samples usingultrafiltration, followed by concentration measurements by absorbancereadings at 280 nm and visual inspection of the samples.

Materials and Methods

The Z-ABD-Z polypeptides ZAZ3363 (SEQ ID NO:1244), ZAZ3364 (SEQ IDNO:1245) and ZAZ3422 (SEQ ID NO:1247) were diluted in PBS, pH 7.4, to2.5 mg/ml. 12 Amicon Ultra centrifugal filter units with a cut-off of 3kDa (Millipore, cat. no. UFC800324) were prerinsed with PBS bycentrifugation at 4000 g for 10 min in a swinging bucket rotorcentrifuge. The concentrators were emptied, and 4 ml of each Z-ABD-Zpolypeptide were added to a first set of three different centrifugalfilter units. Centrifugation was performed at 4000 g, 20° C., for 13-16min, resulting in approximately 1 ml concentrate. A 20 μl sample wasremoved from each concentrate (UF sample 1) for further analysis and therest of the sample volumes were transferred to a second set of threecentrifugal filter units. The centrifugation and sample removal wererepeated three times with spinning times of 8-10 min, 7 min and 13 min,respectively, (UF samples 2, 3 and 4, respectively). Absorbance readingswere performed using a NanoDrop® ND-1000 Spectrophotometer and bydiluting UF samples 1-4 in PBS 3, 6, 12 and 24 times, respectively. Theconcentrations were calculated using the theoretical extinctioncoefficient 1 Abs280=0.612 mg/ml (the same for all three Z-ABD-Zpolypeptides). Absorbance readings of the undiluted filtrates were alsoperformed.

Results

Concentrations determined by absorbance readings at 280 nm after eachcentrifugal step are shown in Table 15. No aggregates were detected byvisual inspection of the concentrates. Thus, the solubility of ZAZ3363,ZAZ3364 and ZAZ3422 were determined to be at least 60 mg/ml in PBS, pH7.4. Absorbance readings of the undiluted filtrates showedconcentrations very close to 0 mg/ml.

TABLE 15 Concentration after consecutive concentration of Z-ABD-Zsamples Total time of Z-ABD-Z SEQ Concentration (mg/ml) centrifugationpolypeptide ID NO UF1 UF2 UF3 UF4 (min) ZAZ3363 1244 9.6 21 36 64 44ZAZ3364 1245 8.2 21 38 60 43 ZAZ3322 1247 9.3 20 35 64 44

Example 11 Biacore Binding Cross-Species Analysis Materials and Methods

The interaction of the Z-ABD-Z polypeptides ZAZ3363 (SEQ ID NO:1244),ZAZ3364 (SEQ ID NO:1245) and ZAZ3365 (SEQ ID NO:1246) with hIL-17A andcynomolgus monkey IL-17A (cIL-17A, SEQ ID NO:1229, Evitria, customorder) as well as the interaction of ZAZ3220 (SEQ ID NO:1236) withhIL-17A and rhesus monkey IL-17A (rmIL-17-A, SEQ ID NO:1230, Cusabio,cat. no. CSB-EP011597MOV) were analyzed in a Biacore 2000. HSA wasimmobilized in the flow cell on the carboxylated dextran layer of a CM5chip surface. The immobilization was performed using amine couplingchemistry according to the manufacturer's protocol and using HBS-EP asrunning buffer. The HSA immobilization levels on the surfaces were 953RU (used for ZAZ3363, ZAZ3364 and ZAZ3365) and 493 RU (used forZAZ3220), respectively. One flow cell surface on the chip was activatedand deactivated for use as blank during analyte injections. In thebinding experiments, HBS-EP was used as running buffer and the flow ratewas 30 μl/min. ZAZ3363, ZAZ3364 and ZAZ3365 were diluted in HBS-EPrunning buffer to a final concentration of 200 nM and injected for 5min, followed by injections of the IL-17A variants. ZAZ3220 was dilutedin HBS-EP running buffer to a final concentration of 500 nM and injectedfor 4 min followed by injections of the IL-17A variants. hIL-17A andcIL-17A were each diluted in HBS-EP running buffer to finalconcentrations of 2.5, 10 and 40 nM and injected for 5 min over surfaceswith ZAZ3363, ZAZ3364 and ZAZ3365 captured on HSA. After 10 min ofdissociation, the surface was regenerated with two injections of 10 mMHCl. hIL-17A and rmIL-17A were diluted in HBS-EP running buffer to finalconcentrations of 0.1, 0.3, 0.9, 2.7, and 8.1 nM and 2.7, 8.1, 24.3, 72and 216 nM, respectively, and injected for 6 min over surfaces withZAZ3220 captured on HSA. After 5 min of dissociation, the surface wasregenerated with two injections of 10 mM HCl. The results were analyzedusing the BiaEvaluation software.

Results

Binding of hIL-17A and cIL-17A to ZAZ3363, ZAZ3364 and ZAZ3365, andhIL-17A and rmIL-17A to ZAZ3220 were tested in a Biacore instrument byinjecting the Z-ABD-Z polypeptides over a surface containing HSAfollowed by injections of the IL-17A variants. All tested Z variantsshowed binding to the tested IL-17A variants, i.e. ZAZ3363, ZAZ3364 andZAZ3365 showed binding to human and cynomolgus monkey IL-17A (FIGS.6A-6C) and ZAZ3220 to human and rhesus monkey IL-17A.

Example 12 Biacore Binding Specificity Analysis

In this Example, the specificities of two Z-ABD-Z polypeptides weretested by analysing their potential interaction with a set of proteinsof the IL-17 family, with other interleukins and with other abundantplasma proteins. The same set of analyte proteins were also analyzed fortheir potential interaction with the ABD variant PEP07843.

Materials and Methods

The interaction of ZAZ3363 (SEQ ID NO:1244) or ZAZ3422 (SEQ ID NO:1247)with panels of 24 or 12 different proteins (specified in Table 16),respectively, were analyzed in a Biacore 2000 instrument. HSA and theABD variant PEP07843 (SEQ ID NO:1225, corresponding to PP013 (SEQ IDNO:1224) with an N-terminal extension of GSS) were immobilized on a CM5chip surface and a blank surface was prepared as described in Example11. The ligand immobilization levels on the surfaces were 1315 RU and 99RU of HSA and PEP07843, respectively, for the chip used in ZAZ3363 runs,and 791 RU and 100 RU of HSA and PEP07843, respectively, for the chipused in ZAZ3422 runs. In the binding experiment, HBS-EP was used asrunning buffer and the flow rate was 30 μl/min. ZAZ3363 and ZAZ3422 werediluted in HBS-EP or HBS-EP supplemented with 500 mM NaCl to a finalconcentration of 200 nM and injected for 7 min followed by injections ofthe analytes. The analytes, i.e. the panels of 24 or 12 differentproteins, were each diluted in HBS-EP or HBS-EP supplemented with 500 mMNaCl to final concentrations of from 0.4 to 250 nM and injected for 5min. After 7 (ZAZ3363) or 10 (ZAZ3422) min of dissociation, the surfaceswere regenerated with four (ZAZ3363) or five (ZAZ3422) injections of 10mM NaOH and two injections of 10 mM HCl. The results were analyzed usingthe BiaEvaluation software.

TABLE 16 Analyte proteins tested with ZAZ3363 and ZAZ3422 Secondinjection First injection Analyte ZAZ3363 ZAZ3422 conc. Buffer for (200nM) (200 nM) Analyte protein Cat. No. (nM) sample dilution Yes YeshIL-17A¹ 200-17 2, 10, 50 HBS-EP and HBS-EP + NaCl Yes Yes hIL-17A/F²5194-IL-025/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-17B² 1248-IB-025/CF 50,250 HBS-EP + NaCl Yes Yes hIL-17C² 1234-IL-025/CF 50, 250 HBS-EP Yes YeshIL-17D² 1504-IL-025/CF 50, 250 HBS-EP + NaCl Yes Yes hIL-17E²1258-IL-025/CF 50, 250 HBS-EP Yes Yes hIL-17F² 1335-IL/CF 50, 250HBS-EP + NaCl Yes Yes hIL-1beta¹ 200-1B 50, 250 HBS-EP Yes Yes hIL-6²206IL/CF 50, 250 HBS-EP Yes Yes hIL23² 1290-IL 50, 250 HBS-EP Yes YeshGM-CSF² 215-GM/CF 50, 250 HBS-EP Yes Yes IgG ATC L04AC07 50, 250 HBS-EP(RoActemra)³ Yes No IgA⁴ P80-102 50, 250 HBS-EP Yes No IL-17RA² 177-IR50, 250 HBS-EP Yes No IL-1R1² 269-1R/CF 50, 250 HBS-EP Yes NoAlpha-2-HS- PRO-1644 50, 250 HBS-EP glycoprotein (AHSG Human HEK)⁵ YesNo Haptoglobin PRO-567 50, 250 HBS-EP Human (seems to be beta chain)⁵Yes No Alpha-1- PRO-529 50, 250 HBS-EP antitrypsin (SERPINA1 Human)⁵ YesNo Human a-2 10952-H08B 50, 250 HBS-EP macroglobulin⁶ Yes No Human10870-H08H 50, 250 HBS-EP Hemopexin/ HPX Protein⁶ Yes No AMBP/Alpha 113141-H08H1 50, 250 HBS-EP microglobulin⁶ Yes No Beta-2- 11976-H08H 50,250 HBS-EP microglobulin/ B2M⁶ Yes No Transthyretin/ 12091-H08H 50, 250HBS-EP TTR/ Prealbumin/ PALB Protein⁶ Yes No holo-Transferrin⁷ T4132 50,250 HBS-EP Yes No IL-17RA² 177-IR 50, 250 HBS-EP Yes No IL-1R1²269-1R/CF 50, 250 HBS-EP Suppliers: ¹Peprotech; ²R&D Systems;³Roche/Apoteket AB; ⁴Bethyl; ⁵ProSpec; ⁶Sino Biological Inc; ⁷Sigma

Results

The specificity of ZAZ3363 and ZAZ3422 was tested in a Biacoreinstrument by investigating their interaction with 24 or 12 analyteproteins, respectively. The Z-ABD-Z polypeptides were injected oversurfaces containing HSA followed by injection of the analyte proteins.The only interactions detected for ZAZ3363 and ZAZ3422 were strongbinding by hIL-17A and weaker binding by hIL-17A/F, as expected and inline with the results presented in Example 8. The analyte proteins werealso assessed for their potential interaction with the ABD variantPEP07843, but no interactions were detected. Thus, the Z-ABD-Zpolypeptides appear to be highly specific.

Example 13 Kinetic Measurements of Z-ABD-Z, IL-17A and HSA ComplexesUsing KinExA®

Technical limitations of SPR to accurately determine kinetic parametersfor high affinity interactions and the higher complexity of determiningthe affinity between two dimeric targets by SPR warranted the use ofKinetic Exclusion Assay (KinExA®) technology to further analyze thebinding of Z-ABD-Z polypeptides in complex with HSA to IL-17A, as wellas the binding of Z-ABD-Z polypeptides alone, or in complex with IL-17A,to HSA. The KinExA® measures the equilibrium binding affinity andkinetics between unmodified molecules in solution phase (Darling andBrault, 2004. Assay and Drug Dev Tech 2(6):647-657)

Materials and Methods

The Z-ABD-Z polypeptides ZAZ3220 (SEQ ID NO:1236), ZAZ3363 (SEQ IDNO:1244) and ZAZ3422 (SEQ ID NO:1247), as well as HSA (Novozymes, cat.no. 230-005), human IL-17A (Peprotech, cat. no. 200-17), biotinylated(as described in Example 1) human IL-17A, mouse monoclonal anti-HSAantibody (Abcam, cat. no. 10241) and an in house produced goatpolyclonal anti-Z antibody were sent to Sapidyne Instruments Inc (Boise,Id., USA) who performed the KinExA® mesurements and analysis.

Binding of Z-ABD-Z/HSA to IL-17A: For determination of the affinity,i.e. K_(D), the respective Z-ABD-Z polypeptide, in complex with HSA,were used as a constant binding partner (CBP) and IL-17A was used astitrant. Data analysis was performed using the KinExA® Pro software andapplying a least squares analysis to fit the optimal solutions for theK_(D) and the Active Binding site Concentration (ABC) to a curverepresentative of a 1:1 reversible bi-molecular interaction.

For determination of k_(a), the direct binding curve analysis wasapplied, using the same immobilized IL-17A as the capture reagent forkinetic experiments as for equilibrium experiments. The amount of freeZ-ABD-Z/HSA in the sample was measured pre-equilibrium, yielding datapoints that monitored the decrease in free Z-ABD-Z/HSA as the samplemoved toward equilibrium.

Binding of Z-ABD-Z and Z-ABD-Z/IL-17A to HSA: The Z-ABD-Z polypeptideZAZ3363 was further analyzed for binding to HSA in the presence orabsence of IL-17A. For determination of K_(D), ZAZ3363, free or incomplex with IL-17A, was used as a constant binding partner (CBP) andHSA was used as titrant. Data analysis was performed using the KinExA®Pro software as described above.

For determination of k_(a), the direct binding curve analysis wasapplied, using the same immobilized HSA as the capture reagent forkinetic experiments as for equilibrium experiments. The amount ofZAZ3363/IL-17A, in the sample was measured pre-equilibrium, yieldingdata points that monitored the decrease in free ZAZ3363/IL-17A as thesample moved toward equilibrium.

Results

In a first set of KinExA® measurements, the Z-ADB-Z polypeptidesZAZ3220, ZAZ3363 and ZAZ3422, respectively, in complex with HSA wereshown to bind IL-17A with an exceptionally high affinity, with K_(D)values in the subpicomolar to femtomolar range. The calculated kineticparameters when assuming a monovalent binding between these Z-ABD-Zpolypeptides and dimeric IL-17A are shown in Table 17.

In a second set of KinExA® measurements, the interaction betweenZAZ3363, free or in complex with IL-17A, and HSA was measured. Thecalculated kinetic parameters from these analyses are shown in Table 18.The affinity of ZAZ3363 for HSA was not significantly affected by thepresence of IL-17A.

To summarize, measurements using KinExA® technology indicated anexceptionally high affinity of Z-ABD-Z polypeptides for IL-17A. Themeasured K_(D) of <0.33 μM is superior to the reported affinities fortwo of the clinically most advanced comparators secukinumab (K_(D)=122μM; WO2006/013107 and WO2012/125680) and ixekizumab (K_(D)=2 μM;WO2007/070750). In addition, simultaneous binding to both IL-17A andalbumin was demonstrated, i.e. both binding functions are intact in theZ-ABD-Z fusion protein.

TABLE 17 Kinetic parameters for Z-ABD-Z polypeptides binding to IL-17ASEQ ka^(a) [*] kd K_(D) [*] CBP ID NO (M⁻¹s⁻¹) (s⁻¹) (M) ZAZ3220/HSA1236 1.35 × 10⁷ 1.51 × 10⁻⁷ <6.5 × 10⁻¹⁴ [1.25 × 10⁷- [**] 1.46 × 10⁷]ZAZ3363/HSA 1244 4.78 × 10⁶ 1.55 × 10⁻⁶ 3.23 × 10⁻¹³ [5.64 × 10⁶- [1.54× 10⁻¹³- 4.04 × 10⁶] 5.54 × 10⁻¹³] ZAZ3422/HSA 1247 7.80 × 10⁶ 6.08 ×10⁻⁷ 7.85 × 10⁻¹⁴ [6.37 × 10⁶- [1.63 × 10⁻¹⁴- 9.56 × 10⁶] 1.73 × 10⁻¹³][*] 95% confidence interval [**] 95% confidence interval for K_(D) wasnot resolved

TABLE 18 Kinetic parameters for a Z-ABD-Z polypeptide binding to HSA SEQka [*] kd K_(D) [*] CBP ID NO (M⁻¹s⁻¹) (s⁻¹) (M) ZAZ3363 1244 n.d. n.d.4.85 × 10⁻¹¹ [3.23 × 10⁻¹¹- 6.87 × 10⁻¹¹] ZAZ3363/IL-17A 1244 1.21 × 10⁶3.56 × 10⁻⁵ 2.94 × 10⁻¹¹ [9.35 × 10⁵- [1.72 × 10⁻¹¹- 1.50 × 10⁶] 4.64 ×10⁻¹¹] [*] 95% confidence interval

Example 14 Characterization of Z-ABD-Z Polypeptides in the NHDF AssayMaterials and Methods

Blocking of IL-17A induced IL-6 production in NHDF assay: NHDF cells(Lonza, cat. no. CC-2511) were cultured in fibroblast basal medium(Lonza, cat. no CC-3132) supplied with growth promoting factors (Lonza,cat. no. CC-5034). On the day before the experiment, 5000 cells per wellwere seeded into half area 96 well culture plates (Greiner, cat. no.675180) in 100 μl. On the day of the experiment, dilutions of IL-17Aspecific Z-ABD-Z polypeptides with different linker lengths between theZ and ABD moieties (ZAZ3174 (SEQ ID NO:1233), ZAZ3175 (SEQ ID NO:1234),ZAZ3176 (SEQ ID NO:1235), ZAZ3220 (SEQ ID NO:1236), ZAZ3221 (SEQ IDNO:1237), ZAZ3234 (SEQ ID NO:1238), ZAZ3235 (SEQ ID NO:1239), ZAZ3236(SEQ ID NO:1240), ZAZ3237 (SEQ ID NO:1241), ZAZ3269 (SEQ ID NO:1242) andZAZ3270 (SEQ ID NO:1243)) were prepared in a separate 96-well plate. TheZ-ABD-Z polypeptides were titrated in three-fold steps from 190 nM to0.003 nM in medium containing 0.9 nM hIL-17A and 8 μM HSA. A standardIL-17A curve was also prepared (6.2-0.0001 nM), as well as controlscontaining medium with 0.9 nM IL-17A or medium alone. The medium in theplate with NHDF cells cultured overnight was discarded and 100 μl/wellof the sample was transferred to the cell plate, which was placed in anincubator at 37° C. for 18-24 h. The next day, the IL-6 content insupernatants was quantified using IL-6 specific ELISA as described inExample 3. Additional Z-ABD-Z polypeptides (ZAZ3363 (SEQ ID NO:1244),ZAZ3364 (SEQ ID NO:1245), ZAZ3365 (SEQ ID NO:1246) and ZAZ3422 (SEQ IDNO:1247)) were analyzed in subsequent NHDF assays essentially asdescribed above, but using an NHDF cell line from ATCC (cat. no.PSC-201-012). The blocking capacities of these Z-ABD-Z polypeptides werealso analyzed after incubation of the polypeptides at 40° C. for 2 and 4weeks.

Results

Z-ABD-Z polypeptides were investigated for their capacity to blockIL-17A induced IL-6 production in the NHDF assay. First, it was seenthat the Z-ABD-Z format increased the blocking capacity significantlycompared to the monomeric His₆-Z format, as shown in FIG. 7A whereinhibitory curves for His₆-Z10241 and ZAZ3220 (comprising Z10241) aredisplayed. The shape of the curve for ZAZ3220 suggests that the limit ofdetection for this cell assay is reached, with a one-to-one inhibitioneffect of the ZAZ3220, and it is likely that the Z-ABD-Z polypeptide hasan even better inhibitory effect than can be appreciated from thisexperiment. It is contemplated that the potency of ZAZ3220 is increasedto the assay limit due to the strong avidity effect obtained by bindingthe homodimeric IL-17A. Second, a comparison of Z-ABD-Z polypeptideswith different linker lengths and comprising the Z variants Z06282 (SEQID NO:1206) and Z12876 (SEQ ID NO:1218) revealed that the length of thelinker had a minor effect on the blocking capacity. A set of Z-ABD-Zpolypeptides comprising Z06282 with different linker lengths isdisplayed in FIG. 7B. Additional Z-ABD-Z polypeptides, ZAZ3363, ZAZ3364,ZAZ3365 and ZAZ3422, were analyzed in subsequent NHDF assays, also afterincubation of the Z-ABD-Z polypeptides at 40° C. for two and four weeks.The IL-17A inhibitory capacity was retained even after four weeks'incubation at 40° C. Calculated IC50 values, reflecting the capacity ofdifferent Z-ABD-Z polypeptides to inhibit IL-17A, are summarized inTable 19. These IC50 values are more than three-fold lower than thereported hIL-6 production neutralizing activity (IC50=2.1±0.1 nM) of theIL-17A inhibiting monoclonal antibody secukinumab, AIN457,(WO2006/013107 and WO2012/125680) currently in clinical development.

TABLE 19 Calculated IC50 values from NHDF assays Incubation Z-ABD-Z SEQat 40° C. IC50 polypeptide ID NO (weeks) (nM) ZAZ3174 1233 n.a. 0.63ZAZ3175 1234 n.a. 0.56 ZAZ3176 1235 n.a. 0.50 ZAZ3220 1236 n.a. 0.27ZAZ3221 1237 n.a. 0.39 ZAZ3234 1238 n.a. 0.66 ZAZ3235 1239 n.a. 0.60ZAZ3236 1240 n.a. 0.54 ZAZ3237 1241 n.a. 0.49 ZAZ3269 1242 n.a. 0.42ZAZ3270 1243 n.a. 0.41 ZAZ3363 1244 0 0.38 ZAZ3363 1244 2 0.25 ZAZ33631244 4 0.49 ZAZ3364 1245 0 0.20 ZAZ3364 1245 2 0.40 ZAZ3364 1245 4 0.47ZAZ3365 1246 0 0.45 ZAZ3365 1246 2 0.34 ZAZ3365 1246 4 0.55 ZAZ3422 12470 0.38 ZAZ3422 1247 2 0.45 ZAZ3422 1247 4 0.42

Example 15 In Vivo Neutralization of hIL-17A

Human IL-17A is able to bind to and stimulate the mouse IL-17 receptor,leading to elevation and subsequent secretion of mouse KC (CXCL1)chemokine.

Materials and Methods

In vivo neutralization of hIL-17A in the KC mouse model: Dose rangingexperiments were performed to identify the optimal dose of human IL-17Afor induction of mouse KC. These experiments revealed that a 150 μg/kgdose of human IL-17A induced a suitable level of KC in mouse serumcollected 2 h after IL-17A administration. ZAZ3174 (SEQ ID NO:1233) andZAZ3220 (SEQ ID NO:1236) were analyzed in this model at two differentoccasions. In the first experiment, ZAZ3174 was administered s.c. tomice at three different doses; 0.25, 2.5 and 25 mg/kg, 9 h prior to asubcutaneous injection of human IL-17A. The mice were sacrificed 2 hpost administration of human IL-17A, and KC levels were determined byELISA using a commercially available kit according to the manufacturer'sinstruction (KC Quantikine; R&D Systems, cat no. D1700). In the secondexperiment, ZAZ3220 was administered s.c. to mice at three differentdoses, 0.05, 0.1, 0.4 mg/kg, 9 h prior to a subcutaneous injection ofhuman IL-17A. The mice were sacrificed 2 h post administration of humanIL-17A, and KC levels were determined by ELISA as above.

Results

In vivo neutralization of hIL-17A in the KC mouse model: The assayedZ-ABD-Z polypeptides block the ability of human IL-17A to stimulate themouse IL-17 receptor, leading to inhibition of an elevation of mouse KC,in a dose dependent manner. ZAZ3174 at a dose of 2.5 mg/kg under theconditions described, blocked the induction of KC completely, as shownin FIG. 8A. The second experiment revealed that the Z-ABD-Z polypeptideZAZ3220, comprising an affinity matured Z variant, blocked the IL-17Ainduced KC-response completely at a dose of 0.4 mg/kg (FIG. 8B). Thiscorresponds to a 6-fold enhanced in vivo blocking effect compared toZAZ3174. 72% inhibition was obtained with 0.1 mg/kg ZAZ3220.

Example 16 In Vivo Pharmacokinetics of Z-ABD-Z Polypeptides in Rats

This Example describes two separate experiments in which thepharmacokinetic parameters in rat were determined for two differentZ-ABD-Z polypeptides following subcutaneous (s.c.) and/or intravenous(i.v.) injections.

Materials and Methods

In a first experiment, ZAZ3220 (SEQ ID NO:1236) formulated in PBS wasadministered i.v. (n=3) to Sprague Dawley (SD) male rats (Charles River,Germany) at a dose of 1.2 mg/kg corresponding to 57 nmol/kg. Bloodsamples were collected from the tail vein of each rat at time points0.08, 8, 24, 48, 72, 120, 168, 240, 336, 408 and 504 h afteradministration.

In a second experiment, ZAZ3363 (SEQ ID NO:1244) formulated in PBS wasadministered i.v. (n=5) or s.c. (n=6) to SD male rats at a dose of 1.2mg/kg corresponding to 64 nmol/kg. Blood samples were collected from thetail vein of each rat at time points 0, 0.08, 0.5, 1, 3, 8, 24, 72, 120,168, 216, 264, 336, 408, 456 and 504 h post i.v. administration or attime points 0, 0.08, 0.5, 1, 3, 8, 24, 72, 120 and 168 h post s.c.administration.

Serum was prepared from the blood samples and stored at −20° C. untilanalysis. Quantification of ZAZ3220 and ZAZ3363 in serum from rats wasperformed using a PK-ELISA.

PK-ELISA: 96-well, half-area plates (50 μl/well) were coated with 4μg/well of goat anti-Z Ig (produced in-house) in coating buffer (Sigma,cat. no. C3041) overnight at 4° C. The next day, the plates were blockedwith PBS+0.5% casein for 1.5 h. Individual rat serum, minimally diluted10× in PBS-casein+10% rat serum pool (assay matrix) was added to theplates and titrated in 2-fold dilution series. Included on one plate wasstandard of each Z-ABD-Z polypeptide (titrated between 300 ng/ml and 3μg/ml) and on each plate four controls of each Z-ABD-Z polypeptidediluted to concentration within the linear range of the assay. Standardand controls were diluted in assay matrix. The diluted standard,controls and samples were prepared in separate plates and transferred tothe coated ELISA plates. The plates were incubated for 1.5 h at RTfollowed by 1.5 h incubation with a custom made detection rabbitanti-ABD Ig (4 μg/ml, CUV002) and 1 h incubation with donkey anti-rabbitIgG-HRP (Jackson Immunoresearch, cat. no. 711-035-152). The reaction wasdeveloped with TMB (Thermo Fisher) and the development reaction wasstopped after 15 min with 2 M H2SO4. The absorbance was read at 450 nmin an ELISA reader (Victor³, Perkin Elmer). The concentrations of therespective Z-ABD-Z variant in samples were calculated from the standardcurve using GraphPad Prism5 and a four parameter logistic (4-PL)curve-fit.

Pharmacokinetic analysis: The pharmacokinetic analysis was based onindividual rat serum concentration versus time. Terminal half-life (T½)and bioavailability was estimated using Microsoft Excel and GraphPadPrism and applying a two compartment model.

Results

Mean serum concentration-time profiles of the Z-ABD-Z polypeptidesZAZ3220 and ZAZ3363 following single administrations of 1.2 mg/kg to SDrats are shown in FIG. 9A and FIG. 9B, respectively, and the calculatedpharmacokinetic parameters using a two-compartment analysis aresummarized in Table 20. The estimated half-life (VA) was approximately49 h for both ZAZ3220 and ZAZ3363 following i.v. administration. The t½for ZAZ3363 administrated s.c. was 45 h and the bioavailability wascalculated to 45%.

TABLE 20 Mean pharmacokinetic parameters of Z-ABD-Z polypeptidesfollowing a single i.v. or s.c. administration to SD ratsPharmakokinetic ZAZ3220 ZAZ3363 ZAZ3363 parameter i.v. i.v. s.c. Cmax(nmol/L) 1905 1870 330 Tmax (h) 0.08 0.08 24 AUC_(0-t) (h*nmol/L) 4636942757 19351 t_(1/2) elimination (h) 49 49 45 MRT (h) 35 33 47 CL(L/h/kg) 0.003 0.003 0.003 F (%) n.a. n.a. 45Cmax: Peak serum concentration; Tmax: Time to reach the peak serumconcentration; AUC_(0-t): Area under the concentration-time curve fromtime zero to last quantifiable concentration; T_(1/2): Half-life; MRT:Mean residence time; CL: Clearance; F: Percentage absolutebioavailability, calculated as F=[(Mean AUC_(s.c.)×Dose_(i.v))/(MeanAUC_(i.v)×Dose_(s.c.))]×100

Example 17 Topical Administration of Z Variant to Eyes of Rabbits

Local administration to the eye is beneficial in ophthalmologicdisorders, e.g. uveitis or dry eye diseases. Topical administrationenables extremely high local drug concentrations with a minimal risk ofsystemic side effects. In this Example, the possibility to topicallyadminister Z variant molecules to eyes was investigated in rabbits. Theconcentration of a Z variant molecule and a control IgG was measured inthe anterior chamber (aqueous humor), in the vitreous humor and in serumafter four repeated topical dosings.

Materials and Methods

Production of Z variant: The Z variant Z10199 (SEQ ID NO:1217) derivedfrom Z06282 (SEQ ID NO:1206) but starting AE was subcloned without anytag but with the C-terminal addition of amino acid residues VD.Expression was performed essentially as described in Example 2, andpurification was carried out by anion exchange and reverse phasechromatography. The sample was buffer exchanged to PBS pH 7.2 andendotoxins were removed using an EndoTrap® red 10 column (Hyglos).

Animal study: 48 nmol (1 drop, 50 μl) of Z10199 and ZAZ3174 (SEQ IDNO:1233), respectively, was administered topically to each eye ofrabbits (n=2 per molecule) at time points 0, 1, 2 and 3 hours. A controlhuman IgG antibody (Xolair; Novartis) was administered in the same wayto two different rabbits. As a negative control, one rabbit wasadministered PBS only. After each administration, the eyelids were heldclosed during approximately 30 s to keep the sample on the cornea. After4 h, vitreous humor, aqueous humor and serum were collected. The sampleswere centrifuged to remove tissue debris and aggregates and the levelsof Z10199, ZAZ3174 and antibody, respectively, were quantified by ELISA.

Quantification by ELISA: The concentration of Z10199 and ZAZ3174,respectively, in the collected samples was analyzed by a sandwich ELISAusing an in-house produced polyclonal goat anti-protein Z immunoglobulinfor capture, an in-house produced polyclonal rabbit anti-protein Zimmunoglobulin as primary antibody and goat anti-rabbit IgG-HRP (Dakocat. no. P0448) as secondary antibody. Detection was performed byincubation with ImmunoPure TMB for 15 min at RT and the reaction wasstopped by addition of 2 M H₂SO₄. Absorption at 450 nm was measured withthe microplate reader Victor³. The concentration of Z10199 wascalculated from a standard curve prepared with the same molecule andusing GraphPad prism5 and a non-linear regression formula.

The concentration of IgG in the collected samples was analyzed by an IgGELISA kit (Abcam 100547) and performed as described by the manufacturer,using a standard curve provided and analysis by non-linear regression asabove.

Results

FIG. 10 shows that both Z10199 and ZAZ3174 penetrated the eye aftertopical administration and was present in aqueous and vitreous humor atlow nM concentrations. In contrast, the control IgG antibody was notdetected in either aqueous or vitreous humor. The serum samples could beregarded as negative for both the Z variant and IgG. Thus, the Z variantmolecules of the present disclosure may be delivered by this alternativeroute of administration, which is not available to antibodies, and alocal effect that avoids systemic exposure may be achieved.

Example 18 Pharmacokinetic Analysis of Duodenal Administration ofZAZ3363 Formulations in Rat Materials and Methods

Test item: ZAZ3363 (SEQ ID NO:1244) was formulated in 1) OAF1: 0.12 Msodium chenodeoxycholate (Sigma, cat. no. C8261) and 0.12 M propylgallate (Sigma, cat. no. P3130), pH 7.4; 2) OAF2: 50 mg/ml sodiumcaprate (Sigma, cat. no. 04151); or 3) 50 mM sodium phosphate (PBS), pH7.0, at a concentration of 50 mg/ml (OAF1 and OAF2) or 100 mg/ml (PBS).

NHDF cell assay: The activity of ZAZ3363 (SEQ ID NO:1244) formulated inOAF1, OAF2 or PBS was verified in the IL-17 dependent NHDF cell assaydescribed in Example 14.

Duodenal administration: Male Sprague Dawley rats (Charles River) wereanesthetized with isofluorane, and a small incision was made to localisethe duodenum. An indwelling catheter (R-DUOD, AgnTho's, Sweden) wasinserted surgically into the duodenum 20 mm from its origin in an areaof minimal vasculature. The catheter was tunnelled subcutaneously to theback of the animals. Abdominal musculature was closed with sutures,while the abdominal skin incision and sub scapular exteriorization sitewas closed with stainless steel wound clips. The animals were left torecover for 5-6 days before being taken to the study. Post-operativeanalgesia was administered s.c. prior to surgery (carprofen 5 mg/kg andbuprenorphine 0.05 mg/kg). Carprofen was administered once daily also onthe two days following surgery. Additional doses of buprenorphine wereadministered when necessary. Antibiotics (enrofloxacin 0.3 mg/ml) wasadministered in the drinking water during the recovery period (3-5days), as well as during the experiment. To mask the bitter taste ofenrofloxacin, one piece of sugar was added to 500 ml of drinking water.To ensure a maintained patency, the duodenal catheter was flushed dailywith sterile water (0.2-0.5 ml).

ZAZ3363 was administered directly into duodenum at a dose of 5.4 μmol/kgbody weight formulated in OAF1 or OAF2 (n=2) or at 10.8 μmol/kgformulated in PBS (n=2) in a volume of 0.5 ml at time-point zero. Bloodsamples were taken under isoflurane anesthesia at 1, 3, 8, 24, 72, 120and 168 h after administration and serum was prepared by standardprocedures. The concentration of ZAZ3363 in serum was measured by thequantitative PK-ELISA described in Example 16, but titrating thestandard of ZAZ3363 between 70 and 1 ng/ml.

Pharmacokinetic analysis: The pharmacokinetic analysis was based onindividual rat serum concentration versus time. Terminal half-life (T¹4)and bioavailability was estimated using Microsoft Excel and GraphPadPrism.

Results

Activity of formulated ZAZ3363: The activity of ZAZ3363 formulated inOAF1 and OAF2, respectively, was compared to formulation in PBS in theNHDF cell assay. FIGS. 11A and 11B shows the titration curves of OAF1compared to PBS formulated ZAZ3363 (FIG. 11A) and OAF2 compared to PBSformulated ZAZ3363 (FIG. 11B). The experiment showed that the activityof ZAZ3363 in the two formulations was identical to PBS, i.e. thebiological activity of ZAZ3363 was not affected by the differentexcipients.

Intraduodenal uptake of ZAZ3363: Intestinal uptake of OAF1, OAF2 and PBSformulated ZAZ3363 was examined in a rat model of intraduodenaladministration (i.d.). The experiment showed an increased uptake withOAF1 and OAF2 formulations compared to PBS (FIG. 12). Thebioavailabilities were 0.2, 0.8 and 0.0007 for OAF1, OAF2 and PBS,respectively, as shown in Table 21. OAF2 formulated ZAZ3363 showed thebest uptake, on average 1160 times better compared to formulation in PBSalthough great individual variation was seen. Formulation of ZAZ3363 inOAF1 was on average 260 times better than formulation in PBS.

TABLE 21 Bioavailability and T½ after intraduodenal administration ofZAZ3363 in three different formulations Formulation T½ (h)Bioavailability (%) PBS 43 0.0007 +/− 0.0002 OAF1 43  0.2 +/− 0.02 OAF250 0.8 +/− 1.2

Example 19 Characterization of Anti-IL-17A/Anti-TNF Complexes in theNHDF Assay Materials and Methods

Production of complexes and control antibody: Two different complexestargeting IL-17A and TNF were constructed, as well as an antibody withaffinity for TNF. The antibody denoted “Ada”, having the same CDRsequences and specificity as the commercially available monoclonalantibody adalimumab, was constructed using the heavy chain (HC) andlight chain (LC) sequences HCAda (SEQ ID NO:1231) and LCAda (SEQ IDNO:1232). The IL-17A targeting Z variant Z14253 (SEQ ID NO:1219) moietywas genetically fused, via a flexible 15 residue (GGGGS)₃ linker, to theC-termini of HC_(Ada) or LC_(Ada), resulting in the complexesHC_(Ada)-Z14253 and LC_(Ada)-Z14253, respectively. A schematic of theconstructed complexes is shown in FIG. 13A. Gene synthesis, cloning,production by transient gene expression in CHO cells and purificationusing Protein A affinity chromatography was performed by Evitria AG(Switzerland).

Blocking of IL-17A induced IL-6 production in NHDF assay: The NHDF assaywas performed essentially as described in Example 14, titrating thestudy samples HC_(Ada)-Z14253 and LC_(Ada)-Z14253 and their comparatorsin three-fold steps from 63 nM to 0.003 nM in a medium containing 0.9 nMrhIL-17A. The comparators were ZAZ3363 (SEQ ID NO:1244), the anti-TNFantibody Ada, as well as a negative control Z variant Z04726 (SEQ IDNO:1223, targeting taq polymerase) recombinantly fused to the ABDvariant PP013 (SEQ ID NO:1224) via a VDSS linker (denoted Z04726-PP013),as described in Example 2. A standard IL-17A curve was also prepared(6.2-0.0001 nM) as well as controls containing medium with 0.9 nM IL-17Aor medium alone. The IL-6 content in supernatants was quantified usingthe IL-6 specific ELISA described in Example 3.

Blocking of TNF or TNF/IL-17A induced IL-8 production in NHDF assay:NHDF (Lonza, cat. no. CC-2511) cells were cultured in fibroblast basalmedium (Lonza, cat. no. CC-3132) supplied with growth promoting factors(Lonza, cat. no CC-5034). On the day before the experiment, 5000 cellsper well were seeded into a half area 96 well culture plates (Greiner,cat. no 675180) in 100 μl. On the day of the experiment, dilutions ofHC_(Ada)-Z14253 and LC_(Ada)-Z14253 and the comparators described abovewere prepared in a separate 96-well plate. The complexes and comparatorswere titrated in three-fold steps from 5 nM to 0.0007 nM in mediumcontaining 0.1 nM human TNF (R&D Systems, cat. no. 2210-TA/CF) oramixture of 0.05 nM and 0.1 nM rhIL-17A. Controls containing medium with0.1 nM TNF or a mixture of 0.05 nM and 0.1 nM rhIL-17A or medium alonewere also prepared. The medium in the plate with the overnight culturedNHDF cells was discarded and 100 μl/well of the sample was transferredto the cell plate. The plate was placed in an incubator at 37° C. for18-24 h. The next day, the IL-8 content in supernatants was quantifiedusing an IL-8 specific ELISA.

IL-8 ELISA: IL-8 was quantified by a DuoSet ELISA kit (R&D systems, cat.no. DY208). Half area plates (Costar, cat. no. 3690) were coated withthe anti-IL-8 capture antibody, 4 μg/ml in PBS, 50 μl/well, overnight at4° C. On the day of the analysis, the plate was rinsed twice in tapwater and then blocked with PBS+1% BSA for 2 h. IL-8 standard (R&DSystems, cat. no. 890806), titrated in a 2-fold dilution series (20-0.01ng/ml) and supernatants from the cell assay plate were added to thecoated ELISA plate (50 μl/well) and incubated for 1.5 h at RT. The platewas washed 4 times in an automated ELISA washer and 20 ng/ml (50μl/well) of biotinylated anti-IL-8 detection antibody was added. Afteranother 1 h incubation, the plate was washed and 50 μl ofstreptavidin-HRP (Thermo Fisher, cat. no. N100) diluted 8000× was addedper well. The plate was developed after one additional hour's incubationand washing, with 50 μl TMB (Thermo Fisher, cat. no 34021) per well, andthe reaction was stopped with 50 μl 2 M H₂SO₄. The absorbances were readin a multi label reader (Victor³, Perkin Elmer).

Results

Blocking of IL-17A induced IL-6 production in NHDF assay: The twocomplexes HC_(Ada)-Z14253 and LC_(Ada)-Z14253 were studied with regardto their capacity to block IL-17A induced IL-6 production in the NHDFassay. Results from the NHDF assay are presented in FIG. 13B. BothHC_(Ada)-Z14253 and LC_(Ada)-Z14253 has a similar capacity to blockIL-17A as ZAZ3363. As expected, no inhibition was seen for Ada or thenegative control Z04726-PP013.

Blocking of TNF or TNF/IL-17A induced IL-8 production in NHDF assay: Thetwo complexes HC_(Ada)-Z14253 and LC_(Ada)-Z14253 were studied withregard to their capacity to block TNF or a mixture of TNF/IL-17A inducedIL-8 production in the NHDF assay. Results from the NHDF assay showedthat HC_(Ada)-Z14253 and LC_(Ada)-Z14253 have similar inhibitoryprofiles as the anti-TNF antibody Ada with regard to specific TNFblocking capacity (FIG. 13C). However, HC_(Ada)-Z14253 andLC_(Ada)-Z14253 had superior inhibitory profiles compared to Ada andZAZ3363 in the combination assay, where the blocking effect to both TNFand IL-17 was investigated (FIG. 13D).

Example 20 In Vivo Pharmacokinetics of Z-ABD-Z Polypeptide in Monkeys

This Example describes a repeated dose study conducted over 10 days incynomolgus monkeys administered with the Z-ABD-Z polypeptide ZAZ3363.The results were used for estimation of the half-life of ZAZ3363 incynomolgus.

Materials and Methods

ZAZ3363 (SEQ ID NO:1244) was administered at 20 mg/kg (n=2; 1 male and 1female) and 40 mg/kg (n=4; 2 male and 2 female) as a short i.v. infusionon days 1, 4, 7 and 10. Plasma samples for determination of ZAZ3363concentration were collected in connection with the first dose on day 1(at time points 0 (pre-dose), 5 min, 0.5, 1, 2, 6, 24 and 48 h afteradministration) and last dose on day 10 (at time points 0 (pre-dose), 5min, 0.5, 1, 2, 6, 24, 48 h, 5, 7, 10, 12, 14 and 21 days afteradministration). Quantification of ZAZ3363 in plasma samples was carriedout by LC-MS/MS based on peptides obtained after tryptic digestion ofZAZ3363. Concentration-time profiles were evaluated using bothnon-compartmental methods with separate analyses for day 1 and day 10and compartmental methods evaluating the data for day 1 and day 10combined for each animal.

Results

Mean plasma concentration-time profiles of ZAZ3363 followingadministration on day 1 and day 10 are shown in FIG. 14A and FIG. 14B,respectively. Despite the fact that ZAZ3363 was not administered as asingle dose with full PK evaluation, the available results aftermultiple dosing allows for a prediction of the concentration-timeprofile after single doses. The plasma concentrations decreased in twophases according to a two-compartment behavior. The t½ of the secondphase estimated from this and a second repeated dose study (data notshown) was approximately 4-7.5 days, which is in agreement with thereported t½ for monkey albumin (Deo et al., 1974, J Nutr 104:858-64). Nosignificant gender differences were observed.

Example 21 Oral Administration of Z-ABD-Z Polypeptide in Dogs Materialsand Methods

Preparation of capsules: ZAZ3363 (SEQ ID NO:1244) was formulated in OAF1(see Example 18) at a concentration of 100 mg/ml. The formulation waslyophilized and filled in hard shell capsules subsequently entericcoated (performed by Catalent Pharma Solutions, Beinheim, France). Eachcapsule contained approximately 25 mg ZAZ3363.

Animal study: The animal study was performed at Huntingdon Life Science(Cambridgeshire, UK). Fasted beagle dogs (n=3; female individuals) eachreceived six capsules containing ZAZ3363 (approximately 150 mg). Serumsamples were taken at 0.5, 1, 2, 4, 6, 8, 12, 24 and 96 h afteradministration.

Quantification: The concentration of ZAZ3363 in serum samples wasquantified using a PK sandwich ELISA essentially as described in Example16, but using an in-house produced monoclonal anti-Z IgG in the firstcoating step.

Results

The individual dog serum concentrations versus time profiles of ZAZ3363are shown in FIG. 15. The results showed intestinal uptake of ZAZ3363 inall three animals, but with some variation between individuals. TheZAZ3363 serum concentration reached a maximum of 2-30 nM. Once in thecirculation, the serum levels of ZAZ3363 remains stable at least up to96 h, which is ascribed the interaction with dog albumin of the ABDmoiety PP013 (SEQ ID NO:1223) within ZAZ3363. The ability of PP013 tobind dog albumin has been demonstrated previously (WO2012/004384).

Itemized Listing of Embodiments

1. IL-17A binding polypeptide, comprising an IL-17A binding motif BM,which motif consists of an amino acid sequence selected from:

-   i) EX₂DX₄AX₆X₇EIX₁₀X₁₁LPNLX₁₆X₁₇X₁₈QX₂₀X₂₁AFIX₂₅X₂₆LX₂₈X₂₉    wherein, independently from each other,

X₂ is selected from A, H, M and Y;

X₄ is selected from A, D, E, F, K, L, M, N, Q, R, S and Y;

X₆ is selected from A, Q and W;

X₇ is selected from F, I, L, M, V, W and Y;

X₁₀ is selected from A and W;

X₁₁ is selected from A, D, E, F, G, L, M, N, Q, S, T and Y;

X₁₆ is selected from N and T;

X₁₇ is selected from H, W and Y;

X₁₈ is selected from A, D, E, H and V;

X₂₀ is selected from A, G, Q, S and W;

X₂₁ is selected from A, D, E, F, H, K, N, R, T, V, W and Y;

X₂₅ is selected from A, D, E, G, H, I, L, M, N, Q, R, S, T and V;

X₂₆ is selected from K and S;

X₂₈ is selected from I, L, N and R; and

X₂₉ is selected from D and R;

and

-   ii) an amino acid sequence which has at least 89% identity to the    sequence defined in i).

2. IL-17A binding polypeptide according to item 1, wherein, in sequencei),

X₂ is selected from A, H and M;

X₄ is selected from A, D, E, F, L, M, N, Q, R and Y;

X₁₁ is selected from A, D, E, F, G, L, M, N, S, T and Y;

X₁₈ is selected from A, D, E and V;

X₂₀ is selected from A, G, Q and W;

X₂₁ is selected from E, F, H, N, R, T, V, W and Y;

X₂₅ is selected from A, D, E, G, H, I, L, N, Q, R, S, T and V; and

X₂₈ is selected from I, N and R.

3. IL-17A binding polypeptide according to item 2, wherein, in sequencei),

X₁₆ is T;

X₁₇ is W;

X₂₁ is selected from E, F, H, W, T and Y;

X₂₅ is selected from A, D, E, G, H, I, L, N, Q, R, S and T;

X₂₆ is K; and

X₂₉ is D.

4. IL-17A binding polypeptide according to any preceding item, whereinsequence i) fulfills at least six of the eleven conditions I-XI:

-   -   I. X₂ is A;    -   II. X₄ is selected from D, E and Q;    -   III. X₆ is A;    -   IV. X₇ is selected from F and V;    -   V. X₁₆ is T;    -   VI. X₁₇ is W;    -   VII. X₁₈ is selected from A and D;    -   VIII. X₂₀ is W;    -   IX. X₂₆ is K;    -   X. X₂₈ is R; and    -   XI. X₂₉ is D.

5. IL-17A binding polypeptide according to item 4, wherein sequence i)fulfills at least seven of the eleven conditions I-XI.

6. IL-17A binding polypeptide according to item 5, wherein sequence i)fulfills at least eight of the eleven conditions I-XI.

7. IL-17A binding polypeptide according to item 6, wherein sequence i)fulfills at least nine of the eleven conditions I-XI.

8. IL-17A binding polypeptide according to item 7, wherein sequence i)fulfills at least ten of the eleven conditions I-XI.

9. IL-17A binding polypeptide according to item 8, wherein sequence i)fulfills all of the eleven conditions I-XI.

10. IL-17A binding polypeptide according to any preceding item, whereinX₂X₆, X₂X₁₀ or X₆X₁₀ is AA.

11. IL-17A binding polypeptide according to any preceding item, whereinX₂X₁₇, X₂X₂₀, X₆X₁₇, X₆X₂₀, X₁₀X₁₇ or X₁₀X₂₀ is AW.

12. IL-17A binding polypeptide according to any preceding item, whereinX₂X₂₈, X₆X₂₈ or X₁₀X₂₈ is AR.

13. IL-17A binding polypeptide according to any preceding item, whereinX₁₇X₂₈ or X₂₀X₂₈ is WR.

14. IL-17A binding polypeptide according to any preceding item, whereinX₁₇X₂₀ is WW.

15. IL-17A binding polypeptide according to any preceding item, whereinsequence i) is the sequence from position 8 to position 36 in a sequenceselected from the group consisting of SEQ ID NO:1-1216.

16. IL-17A binding polypeptide according to item 15, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-66, 1200, 1206 and 1214.

17. IL-17A binding polypeptide according to item 16, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-66.

18. IL-17A binding polypeptide according to item 17, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-35.

19. IL-17A binding polypeptide according to item 18, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-27.

20. IL-17A binding polypeptide according to item 19, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-10.

21. IL-17A binding polypeptide according to item 20, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-7.

22. IL-17A binding polypeptide according to item 21, wherein sequence i)is the sequence from position 8 to position 36 in a sequence selectedfrom the group consisting of SEQ ID NO:1-4.

23. IL-17A binding polypeptide according to item 22, wherein sequence i)is the sequence from position 8 to position 36 in SEQ ID NO:1.

24. IL-17A binding polypeptide according to any preceding item, whereinsaid IL-17A binding motif forms part of a three-helix bundle proteindomain.

25. IL-17A binding polypeptide according to item 24, wherein said IL-17Abinding motif essentially forms part of two helices with aninterconnecting loop, within said three-helix bundle protein domain.

26. IL-17A binding polypeptide according to item 25, wherein saidthree-helix bundle protein domain is selected from bacterial receptordomains.

27. IL-17A binding polypeptide according to item 26, wherein saidthree-helix bundle protein domain is selected from domains of protein Afrom Staphylococcus aureus or derivatives thereof.

28. IL-17A binding polypeptide according to any preceding item, whichcomprises an amino acid sequence binding module, BMod, selected from:

-   iii) K-[BM]-DPSQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;    wherein    -   [BM] is an IL-17A binding motif as defined in any one of items        1-23 provided that X₂₉ is D;

X_(a) is selected from A and S;

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S;

X_(e) is selected from D, E and S;

X_(f) is selected from A and S; and

-   iv) an amino acid sequence which has at least 85% identity to a    sequence defined by iii).

29. IL-17A binding polypeptide according to any one of items 1-27, whichcomprises an amino acid sequence binding module, BMod, selected from

-   v) K-[BM]-QPEQS X_(a)X_(b)LLX_(c) EAKKL X_(d)X_(e)X_(f)Q;    wherein    -   [BM] is an IL-17A binding motif as defined in any one of items        1-23 provided that X₂₉ is R;

X_(a) is selected from A and S;

X_(b) is selected from N and E;

X_(c) is selected from A, S and C;

X_(d) is selected from E, N and S;

X_(e) is selected from D, E and

X_(f) is selected from A and S; and

-   vi) an amino acid sequence which has at least 85% identity to a    sequence defined by v).

30. IL-17A binding polypeptide according to item 28 or 29, wherein X_(a)in sequence iii) or v) is A.

31. IL-17A binding polypeptide according to item 28 or 29, wherein X_(a)in sequence iii) or v) is S.

32. IL-17A binding polypeptide according to any one of items 28-31,wherein X_(b) in sequence iii) or v) is N.

33. IL-17A binding polypeptide according to any one of items 28-31,wherein X_(b) in sequence iii) or v) is E.

34. IL-17A binding polypeptide according to any one of items 28-33,wherein X_(c) in sequence iii) or v) is A.

35. IL-17A binding polypeptide according to any one of items 28-33,wherein X_(c) in sequence iii) or v) is S.

36. IL-17A binding polypeptide according to any one of items 28-33,wherein X_(c) in sequence iii) or v) is C.

37. IL-17A binding polypeptide according to any one of items 28-36,wherein X_(d) in sequence iii) or v) is E.

38. IL-17A binding polypeptide according to any one of items 28-36,wherein X_(d) in sequence iii) or v) is N.

39. IL-17A binding polypeptide according to any one of items 28-36,wherein X_(d) in sequence iii) or v) is S.

40. IL-17A binding polypeptide according to any one of items 28-39,wherein X_(e) in sequence iii) or v) is D.

41. IL-17A binding polypeptide according to any one of items 28-39,wherein X_(e) in sequence iii) or v) is E.

42. IL-17A binding polypeptide according to any one of items 28-39,wherein X_(e) in sequence iii) or v) is S.

43. IL-17A binding polypeptide according to any one of items 37 and39-42, wherein X_(d)X_(e) in sequence iii) or v) is selected from EE,ES, SD, SE and SS.

44. IL-17A binding polypeptide according to item 43, wherein X_(d)X_(e)in sequence iii) or v) is ES.

45. IL-17A binding polypeptide according to item 43, wherein X_(d)X_(e)in sequence iii) or v) is SE.

46. IL-17A binding polypeptide according to item 43, wherein X_(d)X_(e)in sequence iii) or v) is SD.

47. IL-17A binding polypeptide according to any one of items 28-46,wherein X_(f) in sequence iii) or v) is A.

48. IL-17A binding polypeptide according to any one of items 28-46,wherein X_(f) in sequence iii) or v) is S.

49. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is A and X_(f) is A.

50. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A and X_(f) is A.

51. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is C and X_(f) is A.

52. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is S and X_(f) is S.

53. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is S and X_(f) is A.

54. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A and X_(f) is S.

55. IL-17A binding polypeptide according to item 28 or 29, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is C and X_(f) is S.

56. IL-17A binding polypeptide according to item 49, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is A; X_(d)X_(e) isND and X_(f) is A.

57. IL-17A binding polypeptide according to item 50, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A; X_(d)X_(e) isND and X_(f) is A.

58. IL-17A binding polypeptide according to item 51, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is C; X_(d)X_(e) isND and X_(f) is A.

59. IL-17A binding polypeptide according to item 52, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is S, X_(d)X_(e) isND and X_(f) is S.

60. IL-17A binding polypeptide according to item 53, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is S, X_(d)X_(e) isND and X_(f) is A.

61. IL-17A binding polypeptide according to item 55, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is C; X_(d)X_(e) isND and X_(f) is S.

62. IL-17A binding polypeptide according to item 49, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is A; X_(d)X_(e) isSE and X_(f) is A.

63. IL-17A binding polypeptide according to item 50, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A; X_(d)X_(e) isSE and X_(f) is A.

64. IL-17A binding polypeptide according to item 51, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is C; X_(d)X_(e) isSE and X_(f) is A.

65. IL-17A binding polypeptide according to item 52, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is S, X_(d)X_(e) isSE and X_(f) is S.

66. IL-17A binding polypeptide according to item 54, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A; X_(d)X_(e) isSE and X_(f) is S.

67. IL-17A binding polypeptide according to item 55, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is C; X_(d)X_(e) isSE and X_(f) is S.

68. IL-17A binding polypeptide according to item 49, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is A; X_(d)X_(e) isES and X_(f) is A.

69. IL-17A binding polypeptide according to item 50, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A; X_(d)X_(e) isES and X_(f) is A.

70. IL-17A binding polypeptide according to item 51, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is C; X_(d)X_(e) isES and X_(f) is A.

71. IL-17A binding polypeptide according to item 52, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is S, X_(d)X_(e) isES and X_(f) is S.

72. IL-17A binding polypeptide according to item 55, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is C; X_(d)X_(e) isES and X_(f) is S.

73. IL-17A binding polypeptide according to item 49, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is A; X_(d)X_(e) isSD and X_(f) is A.

74. IL-17A binding polypeptide according to item 50, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is A; X_(d)X_(e) isSD and X_(f) is A.

75. IL-17A binding polypeptide according to item 51, wherein, insequence iii) or v), X_(a) is A; X_(b) is N; X_(c) is C; X_(d)X_(e) isSD and X_(f) is A.

76. IL-17A binding polypeptide according to item 52, wherein, insequence iii) or v), X_(a) is S; X_(b) is E; X_(c) is S, X_(d)X_(e) isSD and X_(f) is S.

77. IL-17A binding polypeptide according to item 54, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is A; X_(d)X_(e) isSD and X_(f) is S.

78. IL-17A binding polypeptide according to item 55, wherein, insequence iii) or v), X_(a) is S, X_(b) is E; X_(c) is C; X_(d)X_(e) isSD and X_(f) is S.

79. IL-17A binding polypeptide according to item 28, wherein saidsequence iii) is the sequence from position 7 to position 55 in asequence selected from the group consisting of SEQ ID NO:1-1216.

80. IL-17A binding polypeptide according to item 79, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-66, 1200, 1206 and1214.

81. IL-17A binding polypeptide according to item 80, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-66.

82. IL-17A binding polypeptide according to item 81, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-35.

83. IL-17A binding polypeptide according to item 82, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-27.

84. IL-17A binding polypeptide according to item 83, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-10.

85. IL-17A binding polypeptide according to item 84, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-7.

86. IL-17A binding polypeptide according to item 85, wherein sequenceiii) is the sequence from position 7 to position 55 in a sequenceselected from the group consisting of SEQ ID NO:1-4.

87. IL-17A binding polypeptide according to item 86, wherein sequenceiii) the sequence from position 7 to position 55 in SEQ ID NO:1.

88. IL-17A binding polypeptide according to any preceding item, whichcomprises an amino acid sequence selected from:

-   vii) YA-[BMod]-AP;

wherein [BMod] is an IL-17A binding module as defined in any one ofitems 28-87; and

-   viii) an amino acid sequence which has at least 86% identity to a    sequence defined by vii).

89. IL-17A binding polypeptide according to any one of items 1-87, whichcomprises an amino acid sequence selected from:

-   ix) FA-[BMod]-AP;    -   wherein [BMod] is an IL-17A binding module as defined in any one        of items 28-87; and-   x) an amino acid sequence which has at least 86% identity to a    sequence defined by ix).

90. IL-17A binding polypeptide according to any one of items 1-87, whichcomprises an amino acid sequence selected from:

-   xi) FN-[BMod]-AP;    wherein [BMod] is an IL-17A binding module as defined in any one of    items 28-87; and-   xii) an amino acid sequence which has at least 86% identity to a    sequence defined by xi).

91. IL-17A binding polypeptide according to any preceding item, whichcomprises an amino acid sequence selected from:

SEQ ID NO:  1250 ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK, 1251ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK, 1252ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK, 1253ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK, 1254AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK, 1255VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK, 1256AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK, 1257VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK, 1258VDAKYAK-[BM]-DPSQSSELLAEAKKLNDSQAPK, 1259AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1260AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAP, 1261AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAPK, 1262AEAKYAK-[BM]-DPSQSSELLSEAKKLNDAQAP, 1263AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1264AEAKFAK-[BM]-DPSQSSELLSEAKKLNDSQAP, 1265AEAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK, 1266AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1267AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAP, 1268AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1269AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAP, 1270AEAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK, 1271AEAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK, 1272AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK, 1273AEAKYAK-[BM]-DPSQSSELLSEAKKLESSQAP, 1274AEAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK, 1275AEAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK, 1276AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK, 1277AEAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAP, 1278AEAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK, 1279AEAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK, 1280AEAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK, 1281VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK, 1282VDAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK, 1283VDAKYAK-[BM]-DPSQSSELLAEAKKLSEAQAPK, 1284VDAKYAK-[BM]-QPEQSSELLSEAKKLSESQAPK, 1285VDAKYAK-[BM]-DPSQSSELLSEAKKLESSQAPK, 1286VDAKYAK-[BM]-DPSQSSELLAEAKKLESAQAPK, 1287VDAKYAK-[BM]-QPEQSSELLSEAKKLESSQAPK, 1288VDAKYAK-[BM]-DPSQSSELLSEAKKLSDSQAPK, 1289VDAKYAK-[BM]-DPSQSSELLAEAKKLSDSQAPK, 1290VDAKYAK-[BM]-DPSQSSELLAEAKKLSDAQAPK, 1291VDAKYAK-[BM]-QPEQSSELLSEAKKLSDSQAPK, 1292VDAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK, 1293AEAKYAK-[BM]-DPSQSSELLAEAKKLNKAQAPK, and 1294ADAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,wherein [BM] is an IL-17A binding motif as defined in any one of items1-23.

92. IL-17A binding polypeptide according to any preceding item, whichcomprises an amino acid sequence selected from:

(SEQ ID NO: 1281) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

-   -   wherein [BM] is an IL-17A binding motif as defined in any one of        items 1-23; and

-   xiv) an amino acid sequence which has at least 86% identity to the    sequence defined in xiii).

93. IL-17A binding polypeptide according to item 92, wherein sequencexiii) is selected from SEQ ID NO:1-1216.

94. IL-17A binding polypeptide according to item 93, wherein sequencexiii) is selected from SEQ ID NO:1-66, 1200, 1206 and 1214.

95. IL-17A binding polypeptide according to item 94, wherein sequencexiii) is selected from SEQ ID NO:1-66.

96. IL-17A binding polypeptide according to item 95, wherein sequencexiii) is selected from SEQ ID NO:1-35.

97. IL-17A binding polypeptide according to item 96, wherein sequencexiii) is selected from SEQ ID NO:1-27.

98. IL-17A binding polypeptide according to item 97, wherein sequencexiii) is selected from SEQ ID NO:1-10.

99. IL-17A binding polypeptide according to item 98, wherein sequencexiii) is selected from SEQ ID NO:1-7.

100. IL-17A binding polypeptide according to item 99, wherein sequencexiii) is selected from SEQ ID NO:1-4.

101. IL-17A binding polypeptide according to item 100, wherein sequencexiii) is SEQ ID NO:1.

102. IL-17A binding polypeptide according to any one of items 1-91,which comprises an amino acid sequence selected from:

(SEQ ID NO: 1259) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

-   -   wherein [BM] is an IL-17A binding motif as defined in any one of        items 1-23; and

-   xvi) an amino acid sequence which has at least 86% identity to the    sequence defined in xv).

103. IL-17A binding polypeptide according to item 102, wherein sequencexv) is selected from SEQ ID NO:1217-1222.

104. IL-17A binding polypeptide according to item 103, wherein sequencexv) is selected from SEQ ID NO:1218-1222.

105. IL-17A binding polypeptide according to item 104, wherein sequencexv) is selected from SEQ ID NO:1219-1222.

106. IL-17A binding polypeptide according to item 105, wherein sequencexv) is selected from SEQ ID NO:1219 and SEQ ID NO:1222.

107. IL-17A binding polypeptide according to item 106, wherein sequencexv) is SEQ ID NO:1219.

108. IL-17A binding polypeptide according to any preceding item, whichis capable of binding to IL-17A such that the K_(D) value of theinteraction is at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M, such as atmost 1×10⁻⁸ M, such as at most 1×10⁻⁹ M.

109. IL-17A binding polypeptide according to any preceding item, whichis capable of binding to an IL-17A molecule selected from the groupconsisting of human IL-17A and murine IL-17A.

110. IL-17A binding polypeptide according to item 109, which is capableof binding to human IL-17A.

111. IL-17A binding polypeptide according to item 109, which is capableof binding to murine IL-17A.

112. IL-17A binding polypeptide according to any one of items 109-111,which is capable of binding to human IL-17A and to murine IL-17A.

113. IL-17A binding polypeptide according to any one of items 109, 110and 112, wherein said human IL-17A comprises the amino acid sequence SEQID NO:1226 or an antigenically effective fragment thereof.

114. IL-17A binding polypeptide according to any one of items 109, 111and 112, wherein said murine IL-17A comprises the amino acid sequenceSEQ ID NO:1227 or an antigenically effective fragment thereof.

115. IL-17A binding polypeptide according to any preceding item whichcomprises additional amino acids at the C-terminal and/or N-terminalend.

116. IL-17A binding polypeptide according to item 115, wherein saidadditional amino acid(s) improve(s) and/or simplify/simplifiesproduction, purification, stabilization in vivo or in vitro, coupling ordetection of the polypeptide.

117. IL-17A binding polypeptide according to any preceding item inmultimeric form, comprising at least two IL-17A binding polypeptidemonomer units, whose amino acid sequences may be the same or different.

118. IL-17A binding polypeptide according to item 117, wherein saidIL-17A binding polypeptide monomer units are covalently coupledtogether.

119. IL-17A binding polypeptide according to item 118, wherein theIL-17A binding polypeptide monomer units are expressed as a fusionprotein.

120. IL-17A binding polypeptide according to any one of items 117-119,in dimeric form.

121. Fusion protein or conjugate comprising

-   -   a first moiety consisting of an IL-17A binding polypeptide        according to any preceding item; and    -   a second moiety consisting of a polypeptide having a desired        biological activity.

122. Fusion protein or conjugate according to item 121, wherein saiddesired biological activity is a therapeutic activity.

123. Fusion protein or conjugate according to item 121, wherein saiddesired biological activity is a binding activity.

124. Fusion protein or conjugate according to item 123, wherein saidbinding activity is albumin binding activity which increases in vivohalf-life of the fusion protein or conjugate.

125. Fusion protein or conjugate according to item 124, comprising twoIL-17A binding polypeptides, each as defined in any one of items 1-116,with an albumin binding moiety between them.

126. Fusion protein or conjugate according to item 125, which is capableof binding to IL-17A such that the K_(D) value of the interaction is atmost 1×10⁻¹⁰ M, such as at most 1×10⁻¹¹ M, such as at most 1×10⁻¹² M,such as at most 1×10⁻¹³ M.

127. Fusion protein or conjugate according to any one of items 124-126,wherein said second moiety comprises the albumin binding domain ofstreptococcal protein G or a derivative thereof.

128. Fusion protein or conjugate according to item 123, wherein saidbinding activity acts to inhibit a biological activity.

129. Fusion protein or conjugate according to item 123, wherein saidbinding activity acts to stimulate a biological activity.

130. Fusion protein or conjugate according to item 121, wherein saiddesired biological activity is an enzymatic activity.

131. Fusion protein or conjugate according to item 122, wherein thesecond moiety is a therapeutically active polypeptide.

132. Fusion protein or conjugate according to item 131, wherein thesecond moiety is an immune response modifying agent, for example ananti-inflammatory agent.

133. Fusion protein or conjugate according to any one of items 121-122and 130-132, wherein the second moiety is selected from the groupconsisting of human endogenous enzymes, hormones, growth factors,chemokines, cytokines and lymphokines, and agonists, antagonists andinhibitors thereof.

134. Fusion protein or conjugate according to item 131, wherein thesecond moiety is a toxic compound.

135. Fusion protein or conjugate according to item 123, wherein saidbinding activity is binding to an immune response associated factor, forexample an inflammation-associated factor.

136. Complex, comprising at least one IL-17A binding polypeptideaccording to any one of items 1-117 or at least one fusion protein orconjugate according to any one of items 121-135, and at least oneantibody or an antigen binding fragment thereof.

137. Complex according to item 136, wherein said at least one antibodyor antigen binding fragment thereof is selected from the groupconsisting of full-length antibodies, Fab fragments, Fab′ fragments,F(ab′)₂ fragments, Fc fragments, Fv fragments, single chain Fvfragments, (scFv)₂ and domain antibodies.

138. Complex according to item 137, wherein said at least one antibodyor antigen binding fragment thereof is selected from the groupconsisting of full-length antibodies, Fab fragments and scFv fragments.

139. Complex according to item 138, wherein said at least one antibodyor antigen binding fragment thereof is a full-length antibody.

140. Complex according to any one of items 136-139, wherein saidantibody or antigen binding fragment thereof is a monoclonal antibody oran antigen binding fragment thereof.

141. Complex according to any one of items 136-140, wherein saidantibody or antigen binding fragment thereof is selected from the groupconsisting of human antibodies, humanized antibodies and chimericantibodies, and antigen-binding fragments thereof.

142. Complex according to item 141, wherein said antibody or antigenbinding fragment thereof is a human or humanized antibody, or an antigenbinding fragment thereof.

143. Complex according to any one of items 136-142, wherein saidantibody or antigen binding fragment thereof has affinity for anantigen, such as selected from the group consisting of an antigenassociated with an angiogenesis related disorder and an antigenassociated with the immune response or with a disorder of the immunesystem.

144. Complex according to item 143, wherein said antigen is associatedwith an angiogenesis related disorder.

145. Complex according to item 143, wherein said antigen is associatedwith the immune response or with a disorder of the immune system, forexample associated with inflammation.

146. Complex according to any one of items 136-145, which is a fusionprotein or a conjugate.

147. Complex according to any one of items 136-146, wherein said IL-17Abinding polypeptide is attached to the N-terminus or C-terminus of theheavy chain of said antibody or antigen binding fragment thereof.

148. Complex according to any one of items 136-146, wherein said IL-17Abinding polypeptide is attached to the N-terminus or C-terminus of thelight chain of said antibody or antigen binding fragment thereof.

149. Complex according to any one of items 136-146, wherein said IL-17Abinding polypeptide is attached to the N-terminus and/or C-terminus ofthe light chain and heavy chain of said antibody or antigen bindingfragment thereof.

150. Complex according to any one of items 146-149, which is a fusionprotein.

151. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any preceding item, further comprising at least one linker,for example selected from the group consisting of non-peptidic linkers,flexible amino acid linkers, rigid amino acid linkers and cleavableamino acid linkers.

152. Fusion protein or conjugate according item 151, wherein said linkeris arranged between said first moiety and said second moiety.

153. Complex according to item 151, wherein said linker is arrangedbetween said IL-17A binding polypeptide and said antibody or antigenbinding fragment thereof.

154. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording any one of items 151-153, wherein said linker is a flexiblelinker comprising amino acid residues selected from the group consistingof glycine, serine and threonine.

155. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 154, wherein said linker comprises a sequence with ageneral formula selected from

(G_(n)S_(m))_(p) and (S_(n)G_(m))_(p),

wherein, independently,

n=1-7,

m=0-7,

n+m 8 and

p=1-10.

156. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 155, wherein n=1-5.

157. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 155-156, wherein m=0-5.

158. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 155-157, wherein p=1-5.

159. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 156-158, wherein n=4, m=1 and p=1-4.

160. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 159, wherein said linker comprises a sequence selectedfrom the group consisting of G₄S, (G₄S)₂, (G₄S)₃ and (G₄S)₄.

161. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 160, wherein said linker comprises the sequence G₄S.

162. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 155-159, wherein said general formula isGT(G_(n)S_(m))_(p).

163. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 162, wherein said linker comprises the sequence GTG₄S.

164. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any preceding item, further comprising a label.

165. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to item 164, wherein said label is selected from the groupconsisting of fluorescent dyes and metals, chromophoric dyes,chemiluminescent compounds and bioluminescent proteins, enzymes,radionuclides and particles.

166. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any preceding item, comprising a chelating environmentprovided by a polyaminopolycarboxylate chelator conjugated to the IL-17Abinding polypeptide via a thiol group of a cysteine residue or an aminegroup of a lysine residue.

167. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any preceding item, comprising one or more polyethyleneglycol moieties.

168. Polynucleotide encoding a polypeptide according to any one of items1-163.

169. Expression vector comprising a polynucleotide according to item168.

170. Host cell comprising an expression vector according to item 169.

171. Method of producing a polypeptide according to any one of items1-163, comprising

-   -   culturing a host cell according to item 170 under conditions        permissive of expression of said polypeptide from said        expression vector, and    -   isolating said polypeptide.

172. Composition comprising an IL-17A binding polypeptide, fusionprotein, conjugate or complex according to any one of items 1-167 and atleast one pharmaceutically acceptable excipient or carrier.

173. Composition according to item 172, further comprising at least oneadditional active agent, such as an agent selected from the groupconsisting of therapeutically active polypeptides, immune responsemodifying agents, anti-inflammatory agents and toxic compounds.

174. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 1-167 or a composition according to anyone of items 172-173 for oral, topical, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual or suppository administration, such as for oraladministration or such as for topical administration.

175. IL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 1-167 or a composition according to anyone of items 172-173 for use as a medicament, a diagnostic agent or aprognostic agent.

176. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 175, wherein said polypeptide,fusion protein, conjugate, complex or composition modulates IL-17Afunction in vivo.

177. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to any one of items 175-176, wherein saidpolypeptide, fusion protein, conjugate, complex or composition inhibitsIL-17A signaling.

178. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to any one of items 175-177, wherein saidpolypeptide, fusion protein, conjugate, complex or composition blocksbinding of IL-17A to at least one of its cognate receptors.

179. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to any one of items 175-178, in thetreatment, diagnosis or prognosis of an IL-17A associated condition.

180. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 179, wherein said IL-17Aassociated condition is selected from the group consisting ofinflammatory diseases, autoimmune diseases and cancer.

181. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 180, wherein said IL-17Aassociated condition is selected from the group consisting ofinflammatory diseases and autoimmune diseases.

182. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 181, wherein said IL-17Aassociated condition is selected from the group consisting ofinflammatory conditions, allergic conditions, hypersensitivityreactions, autoimmune diseases, severe infections and transplantrejections.

183. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 182, wherein said IL-17Aassociated condition is selected from the group consisting of rheumatoidarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis,multiple sclerosis, systemic lupus erythematosus, uveitis and dry eyedisease.

184. IL-17A binding polypeptide, fusion protein, conjugate, complex orcomposition for use according to item 183, wherein said IL-17Aassociated condition is psoriasis.

185. IL-17A binding polypeptide, fusion protein, conjugate or complexfor use in prognosis according to item 179, wherein said IL-17Aassociated condition is cancer, such as a cancer selected from the groupconsisting of gastric cancers, colorectal cancers, non-small cell lungcancers, hepatocellular carcinomas and adenocarcinomas.

186. Method of detecting IL-17A, comprising providing a sample suspectedto contain IL-17A, contacting said sample with an IL-17A bindingpolypeptide, fusion protein, conjugate or complex according to any oneof items 1-167 ora composition according to any one of items 172-173,and detecting the binding of the IL-17A binding polypeptide, fusionprotein, conjugate, complex or composition to indicate the presence ofIL-17A in the sample.

187. Method for determining the presence of IL-17A in a subject, themethod comprising the steps:

-   -   contacting the subject, or a sample isolated from the subject,        with an IL-17A binding polypeptide, fusion protein, conjugate or        complex according to any one of items 1-167 ora composition        according to any one of items 172-173, and    -   obtaining a value corresponding to the amount of the IL-17A        binding polypeptide, fusion protein, conjugate, complex or        composition that has bound in said subject or to said sample.

188. Method according to item 187, further comprising a step ofcomparing said value to a reference.

189. Method according to item 187 or 188, wherein said subject is amammalian subject, such as a human subject.

190. Method according to any one of items 187-189, performed in vivo.

191. Method according to any one of items 187-189, performed in vitro.

192. Method of treatment of an IL-17A associated condition, comprisingadministering to a subject in need thereof an effective amount of anIL-17A binding polypeptide, fusion protein, conjugate or complexaccording to any one of items 1-167 or a composition according to anyone of items 172-173.

193. Method according to item 192, wherein said IL-17A associatedcondition is selected from the group consisting of inflammatorydiseases, autoimmune diseases and cancer.

194. Method according to item 193, wherein said IL-17A associatedcondition is selected from the group consisting of inflammatory diseasesand autoimmune diseases.

195. Method according to item 194, wherein said IL-17A associatedcondition is selected from the group consisting of inflammatoryconditions, allergic conditions, hypersensitivity reactions, autoimmunediseases, severe infections and transplant rejections.

196. Method according to item 195, wherein said IL-17A associatedcondition is selected from the group consisting of rheumatoid arthritis,ankylosing spondylitis, psoriatic arthritis, psoriasis, multiplesclerosis, systemic lupus erythematosus, uveitis and dry eye disease.

197. Method according to item 196, wherein said IL-17A associatedcondition is psoriasis.

1. A method of treatment of an IL-17A associated condition, comprisingadministering to a subject in need thereof an effective amount of anIL-17A binding polypeptide, wherein said IL-17A binding polypeptidecomprises an IL-17A binding motif BM, which motif consists of an aminoacid sequence selected from: (SEQ ID NO: 1295)EX₂DX₄AX₆X₇EIX₁₀X₁₁LPNLX₁₆X₁₇X₁₈QX₂₀X₂₁AFIX₂₅X₂₆L X₂₈X₂₉

wherein, independently from each other, X₂ is selected from A, H, M andY; X₄ is selected from A, D, E, F, K, L, M, N, Q, R, S and Y; X₆ isselected from A, Q and W; X₇ is selected from F, I, L, M, V, W and Y;X₁₀ is selected from A and W; X₁₁ is selected from A, D, E, F, G, L, M,N, Q, S, T and Y; X₁₆ is selected from N and T; X₁₇ is selected from H,W and Y; X₁₈ is selected from A, D, E, H and V; X₂₀ is selected from A,G, Q, S and W; X₂₁ is selected from A, D, E, F, H, K, N, R, T, V, W andY; X₂₅ is selected from A, D, E, G, H, I, L, M, N, Q, R, S, T and V; X₂₆is selected from K and S; X₂₈ is selected from I, L, N and R; and X₂₉ isselected from D and R; and ii) an amino acid sequence which has at least89% identity to the sequence defined in i).
 2. The method according toclaim 1, wherein, in sequence i) of the IL-17A binding polypeptide, X₂is selected from A, H and M; X₄ is selected from A, D, E, F, L, M, N, Q,R and Y; X₁₁ is selected from A, D, E, F, G, L, M, N, S, T and Y; X₁₈ isselected from A, D, E and V; X₂₀ is selected from A, G, Q and W; X₂₁ isselected from E, F, H, N, R, T, V, W and Y; X₂₅ is selected from A, D,E, G, H, I, L, N, Q, R, S, T and V; and X₂₈ is selected from I, N and R.3. The method according to claim 2, wherein, in sequence i) of theIL-17A binding polypeptide, X₁₆ is T; X₁₇ is W; X₂₁ is selected from E,F, H, W, T and Y; X₂₅ is selected from A, D, E, G, H, I, L, N, Q, R, Sand T; X₂₆ is K; and X₂₉ is D.
 4. The method according to claim 1,wherein sequence i) of the IL-17A binding polypeptide consists of aminoacids 8 to 36 of any one of SEQ ID NO:1-1216.
 5. The method according toclaim 1, wherein the IL-17A binding polypeptide comprises a bindingmodule, BMod, consisting of an amino acid sequence selected from:(SEQ ID NO: 1296) K-[BM]-DPSQSX_(a)X_(b)LLX_(c)EAKKLX_(d)X_(e)X_(f)Q,

wherein [BM] is the IL-17A binding motif as defined in claim 1 providedthat X₂₉ is D; X_(a) is selected from A and S; X_(b) is selected from Nand E; X_(c) is selected from A, S and C; X_(d) is selected from E, Nand S; X_(e) is selected from D, E and 5, and X_(f) is selected from Aand 5, and iv) an amino acid sequence which has at least 85% identity toa sequence defined by iii).
 6. The method according to claim 5, whereinsequence iii) consists of amino acids 7 to 55 of any one of SEQ IDNO:1-1216.
 7. The method according to claim 5, wherein the IL-17Abinding polypeptide comprises an amino acid sequence selected from: vii)YA-[BMod]-AP (SEQ ID NO:1298), wherein [BMod] is the binding module asdefined in claim 5; and vii) an amino acid sequence which has at least86% identity to a sequence defined by vii).
 8. The method according toclaim 1, wherein the IL-17A binding polypeptide comprises an amino acidsequence selected from: (SEQ ID NO: 1281)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

wherein [BM] is the IL-17A binding motif as defined in claim 1; and xiv)an amino acid sequence which has at least 86% identity to a sequencedefined by xiii).
 9. The method according to claim 8, wherein sequencexiii) is selected from the group consisting of SEQ ID NO:1-1216.
 10. Themethod according to claim 1, wherein the IL-17A binding polypeptidecomprises an amino acid sequence selected from: (SEQ ID NO: 1259)AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK,

wherein [BM] is the IL-17A binding motif as defined in claim 1; and xvi)an amino acid sequence which has at least 86% identity to a sequencedefined by xiii).
 11. The method according to claim 10, wherein sequencexv) is selected from the group consisting of SEQ ID NO:1217-1222. 12.The method according to claim 1, wherein the IL-17A binding polypeptidebinds to IL-17A with a K_(D) value of at most 1×10⁻⁶ M.
 13. The methodaccording to claim 1, wherein the IL-17A binding polypeptide iscomprised in a fusion protein or conjugate comprising a first moietyconsisting of the IL-17A binding polypeptide as defined in claim 1, anda second moiety consisting of a polypeptide having a desired biologicalactivity.
 14. The method according to claim 13, wherein said biologicalactivity is albumin binding activity which increases in vivo half-lifeof the fusion protein or conjugate.
 15. The method according to claim14, wherein said fusion protein or conjugate comprises two IL-17Abinding polypeptides, each as defined in claim 1, with an albuminbinding moiety between them.
 16. The method according to claim 15,wherein said fusion protein or conjugate comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1233-1247. 17.The method according to claim 16, wherein said fusion protein orconjugate comprises the amino acid sequence SEQ ID NO:1244.
 18. Themethod according to claim 1, wherein the IL-17A binding polypeptide iscomprised in a complex, comprising at least one IL-17A bindingpolypeptide as defined in claim 1 or at least one fusion protein orconjugate as defined in claim 13, and at least one antibody or anantigen binding fragment thereof.
 19. The method according to claim 1,wherein the IL-17A binding polypeptide is comprised in a compositioncomprising at least one IL-17A binding polypeptide as defined in claim 1or at least one fusion protein or conjugate as defined in claim 13, andat least one pharmaceutically acceptable excipient or carrier.
 20. Themethod according to claim 1, wherein the IL-17A binding polypeptide iscomprised in a composition comprising at least one complex as defined inclaim 14, and at least one pharmaceutically acceptable excipient orcarrier.
 21. The method according to claim 1, wherein said IL-17Aassociated condition is selected from the group consisting ofinflammatory diseases, autoimmune diseases and cancer.
 22. The methodaccording to claim 21, wherein said IL-17A associated condition isselected from the group consisting of inflammatory diseases andautoimmune diseases.
 23. The method according to claim 1, wherein saidIL-17A associated condition is selected from the group consisting ofinflammatory conditions, allergic conditions, hypersensitivityreactions, autoimmune diseases, severe infections and transplantrejections.
 24. The method according to claim 23, wherein said IL-17Aassociated condition is selected from the group consisting of rheumatoidarthritis, juvenile rheumatoid arthritis, Takayasu's arteritis,spondyloarthropathies, ankylosing spondylitis, psoriatic arthritis,psoriasis, multiple sclerosis, systemic lupus erythematosus, uveitis anddry eye disease.
 25. The method according to claim 24, wherein saidIL-17A associated condition is selected from spondyloarthropathies,ankylosing spondylitis, psoriatic arthritis, psoriasis, Takayasu'sarteritis and uveitis.
 26. The method according to claim 25, whereinsaid IL-17A associated condition is ankylosing spondylitis.
 27. Themethod according to claim 25, wherein said IL-17A associated conditionis psoriatic arthritis.
 28. The method according to claim 25, whereinsaid IL-17A associated condition is psoriasis.
 29. The method accordingto claim 25, wherein said IL-17A associated condition is Takayasu'sarteritis.
 30. The method according to claim 25, wherein said IL-17Aassociated condition is uveitis.
 31. The method according to claim 25,wherein said IL-17A associated condition is a spondyloarthropathy.